Biomedical Engineering Reference
In-Depth Information
Table 1
Scar-less DNA assembly methodologies and protocol design software
Category
Representative methodologies
Protocol design software
Flanking homology
sequence
Quick and Clean cloning
(Chapter
3
) [
21
]
j5 (this chapter) [
17
,
31
]
In-Fusion
®
(Chapter
15
)
Gibthon [
32
]
SLIC (Chapter
2
) [
22
]
GeneDesign [
33
-
35
]
Simple cloning (Chapter
13
)
[
23
]
BioCAD [
7
]
CPEC (Chapter
8
) [
10
,
22
]
In-Fusion
®
Primer Design [
36
]
TPCR (Chapter
7
) [
25
]
GeneArt
®
OligoDesigner [
37
]
SLiCE (Chapter
16
) [
26
]
Gibson [
27
,
28
]
GeneArt
®
seamless/High-Order
DNA assembler [
11
,
29
]
Reiterative recombination [
30
]
Type IIS endonuclease
Golden gate (Chapter
9
)
[
38
-
40
]
j5
FX cloning (Chapter
11
) [
41
]
GeneDesign
BioCAD
Uracil-excision
USER (Chapter
5
) [
42
,
43
]
PHUSER (Chapter
5
)
[
44
,
45
]
GeneDesign
This chapter provides a brief tutorial on how to design
(combinatorial) scar-less DNA assembly protocols using the web-
based software j5 [
17
]. j5 assists biomedical and biotechnological
researchers construct DNA by automating the design of optimized
protocols for fl anking homology sequence as well as type IIS
endonuclease-mediated DNA assembly methodologies (Table
1
).
Unlike any other software tool available today, j5 designs scar-less
combinatorial DNA assembly protocols, performs a cost-benefi t
analysis to identify which portions of an assembly process would be
less expensive to outsource to a DNA synthesis service provider,
and designs hierarchical DNA assembly strategies to mitigate antic-
ipated poor assembly junction sequence performance. Software
integrated with j5 (e.g., DeviceEditor [
18
], VectorEditor [
19
],
and PR-PR [
20
]) add signifi cant value to the j5 design process
through graphical user-interface enhancement and downstream
liquid-handling robotic laboratory automation.
2
Materials
2.1 j5 and
DeviceEditor Software
The j5 and DeviceEditor web-based software described in this
chapter is available to academic, nonprofi t, and government
researchers at no cost on the j5 web-server [
31
]. The j5 and
DeviceEditor software has been exclusively licensed to TeselaGen
Biotechnology, Inc. [
46
] for commercial use and distribution.
