Transposition efficiency is one important criterium
for judging a transposon system. High transposition efficiency
is especially desirable for insertion of primer-binding sites into
large BAC or fosmid clones in vitro in order to determine
its complete sequence. High transposition efficiency is also important
for insertion of a transposon into DNA in a living cell in vivo.
EZ-Tn5 Systems have very high transposition efficiencies for in
vitro insertion, as well as for in vivo insertion of
an EZ-Tn5 Transposome™ Complex, the synaptic complex formed
between an EZ-Tn5 Transposase and an EZ-Tn5 Transposon. Although
Epicentre's HyperMu™ MuA Transposase is at least 50 times
more active for in vitro transposition than the MuA transposase
from other suppliers and almost as active as the EZ-Tn5 System in
vitro, the HyperMu Transposome™ Complex generates 10-100
times fewer insertions than the EZ-Tn5 System in vivo using E.
coli. Tn7-based transposon systems are quite efficient for in
vitro insertion, but have undetectable activity as a synaptic
complex, probably because the active Tn7 transposase consists of
3 proteins, which may be difficult to electroporate into the cell
as a complex. The Ty1 system has very low transposition efficiency.
For most applications, it is also important that transposition
is random. Based on our experience with different systems, we believe
that the EZ-Tn5 System is the most random. However, all four transposon
systems seem to be sufficiently random for most applications, including
for determining the complete sequence of BAC or fosmid clones.
Although Tn7 insertion is highly specific in the presence of the
TsnE Protein, target specificity is not observed using a transposase
consisting of only the TsnABC proteins. A study comparing all systems
using transposons with the same selectable marker and other identical
transposon features into identical target sequences has not been
published. Comparisons using different selectable markers or other
transposon features, or different targets are flawed because variations
in transposon features and in the DNA target chosen can affect
which insertions are viable and selected for analysis.
In general, it is desirable that the length of the transposase
recognition sequences in the "transposon ends" are short
when primer binding sites in the transposon are used for sequencing.
Shorter transposon ends maximize sequence data obtained from the
target DNA and minimize undesired sequence data from the end of
the primer to the end of the transposon.
(fmoles) ||Template Size (insert + vector) in Kilobases |
|1 kb ||3 kb ||5 kb ||7 kb || 9 kb |
| 25 |
|17 ng ||50 ||83 ||120 ||150 |
|50 || 33 ||100 ||165 ||230 ||300 |
| 75 |
| 50 ||150 ||250 ||350 ||450 |
| 100 ||66 ||200 ||330 ||460 |
| 600 |
| 150 ||100 ||300 ||500 ||700 ||900 |
| 200 ||135 ||400 ||660 ||930 ||1200 |
|250 ||165 ||500 ||830 ||1200 ||1550 |
| 300 || 200 ||600 ||1000 ||1400 ||1800 |