Download Design Genes with Ease Using In-Fusion® Cloning

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Design Genes with Ease
Using In-Fusion® Cloning
The work described in this article was performed at Harvard Medical
School1 by B. Zhu, G. Cai, E.O. Hall, and G.J. Freeman and originally
published in BioTechniques (1).
1 Dana-Farber Cancer Institute, Department of Medicine, Harvard Medical School,
Boston, MA 02115, USA
This article has been adapted from the original publication.
In-Fusion can join any two pieces of DNA that contain a 15 bp overlap at their ends.
The result is equivalent to a recombination event across the 15 bp overlap.
The 15 bp overlap can be engineered by inclusion in forward and reverse primers
used to PCR-amplify two segments of DNA. Originally described for inserting
one piece of DNA into a restriction enzyme-digested plasmid, we have found
that In-Fusion can efficiently join up to four pieces of DNA in a single reaction.
We used this approach to construct seamless fusion proteins and modular
vectors with readily interchangeable segments. In-Fusion-Ready DNA modules
can be pieced together simultaneously without restriction digestion, regardless
of sequence and size, thereby opening up new cloning possibilities.
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DNA constructs are typically created by ligating two different DNA fragments
that have been digested with restriction enzymes containing complementary
restriction sites. Cloning options are often limited by the lack of available unique
sites in the vector and gene of interest. In contrast, In-Fusion is an enabling
technology that can join any two pieces of DNA that contain 15 bp of shared
identity at their ends, without the need for any restriction enzyme digestion.
The 15 bp overlap can be included in the primers used to PCR-amplify a segment
of DNA. Moreover, the pieces of DNA can be generated either by PCR or by restriction
digestion of plasmid DNA, regardless of the types of ends (sticky or blunt) produced.
The In-Fusion mechanism is ligase-independent and uses the unique properties
of poxvirus DNA polymerase (2–4). When incubated with linear duplex DNAs
with identical ends in the presence of Mg2+ and low concentrations of dNTP,
the 3'–5' proofreading activity of poxvirus DNA polymerase progressively
removes nucleotides from the 3' end. This exposes complementary regions
on the DNA fragments that can then spontaneously anneal through base
pairing. This results in joined molecules containing a hybrid region flanked
by nicks, 1–5 nucleotide gaps, or short overhangs. The annealed structures
are moderately long-lived because the poxvirus DNA polymerase has a lower
affinity for nicked or gapped DNA ends than for duplex ends. Introduction
of this annealed DNA into Escherichia coli repairs any single-stranded gaps.
In this study, we explore the unique end-joining capabilities of In-Fusion to combine
not only two, but four fragments of DNA in a single, one-step reaction to develop
seamless fusion proteins and modular expression vectors.
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Construct Design
DNA sequence segments encoding the extracellular domain of murine (m) PD-L2 (5), the crystallizable fragment (Fc)
of murine immunoglobulin G2a (IgG2a) (5), and the murine IgA tailpiece (6, 7), were assembled in silico with a mammalian
expression vector using the Sequencher program (Gene Codes). The goal was to create a murine dodecameric
immunoglobulin fusion protein (6, 7). Sense and antisense PCR primers were designed containing 15 bp of sequence
which overlapped with the adjacent segment of the construct, as well as 20–30 bp of segment-specific sequence (see
Figure 1 and Table I). The multiple pieces of DNA were joined seamlessly because the In-Fusion Enzyme does not
add any additional nucleotides during the cloning reaction.
All DNA fragments shown here were PCR-amplified from appropriate templates using overlapping primers that
were designed as described in the In-Fusion Kit User Manual. Clontech® also provides an online tool for primer
design.
Fragments generated by PCR were amplified by a high-fidelity, hot start polymerase, gel-purified, and quantified
by absorbance. The vectors used as backbones in this study were linearized using NcoI and SalI restriction enzyme
sites. However, vector backbones with the desired 15 bp overhangs can also be generated by PCR using a highfidelity DNA polymerase (data not shown).
In-Fusion reactions were set up according to the Clontech In-Fusion Kit User Manual. Between 25–100 ng of vector
was mixed at a ratio of 1 mole vector to 2 moles of each additional DNA segment in 10 µl of water and added to one
well containing the lyophilized In-Fusion reaction mix. After incubation, the In-Fusion reaction was diluted with TE
and transformed into chemically competent E. coli according to the In-Fusion User Manual, miniprepped, and characterized by restriction enzyme digestion and sequencing.
PD-L2
extracellular
domain
Table I: Primers for PCR Amplification of the PD-L2
Extracellular Domain, IgG2a Fc, and IgA Tailpiece
PD-L2 Extracellular Domain with 15 bp Overlaps to
NcoI-Digested Vector and IgG2a Fc, 693 bp PCR Product
Pr
o
m
NcoI
er
ot
mlgG2a Fc
Sense (NcoI site is underlined)
5'-TTCAAATCCACCATGGAGACAGACACACTCCTGCTAT-3'
mlgA tailpiece
Antisense
5'-CTTGATTGTGGGCCCTCTGGGGACTTTGGGTTCCATCCGA-3'
IgG2a Fc, 678 bp PCR Product
)
(A
SalI
Po
ly
Expression Vector
Sense
5'-GGGCCCACAATCAAGCCCTGTCCTC-3'
Antisense
5'-CCGGGAGAAGCTCTTAGTC-3'
IgA Tailpiece with 15 bp Overlaps to IgG2a Fc
and SalI-Digested Vector, 100 bp PCR Product
Sense
5'-AAGAGCTTCTCCCGGCTGTCGGGTAAACCCACCAATGTCAG-3'
Figure 1. Seamless construction of a three-domain immunoglobulin
fusion protein with a four-piece In-Fusion reaction. Segments
were generated by PCR using primers that contained a 15 bp
overlap with the adjacent segment, and 20–30 bp of segmentspecific sequence. Colored regions indicate overlap regions
with 15 bp of identity. Arrows indicate PCR primers. The segments
were joined to a NcoI-SalI-digested expression vector in a ligasefree In-Fusion reaction.
Antisense (SalI site is underlined)
5'-AGTAACGTTAGTCGACTCAGTAGCAGATGCCATCTC-3'
Overlaps are colored to match Figure 1.
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Multiple Fragment Cloning Results
A major limitation of standard cloning approaches is the addition of unwanted amino acids that are encoded by the
restriction enzyme sites used to join the DNA ends. This is particularly detrimental for fusion proteins and recombinant
antibodies, since the undesired amino acids may perturb structure, reduce expression, or be antigenic. We generated
a seamless murine PD-L2-IgG2a Fc-IgA tailpiece fusion protein by joining four pieces of DNA encoding the PD-L2
extracellular domain, IgG2a Fc domain, IgA tailpiece, and an expression vector containing a DHFR selection cassette.
NcoI and SalI sites were used to linearize the vector and were therefore incorporated into the 5' and 3' segment
fragments as part of the 15 bp complementary overhang with the linearized backbone (see Table I).
No restriction sites were incorporated between the fusion protein domains. Since no restriction digestion of inserts
is needed when using In-Fusion technology, the presence of NcoI and SalI restriction sites within the amplified
fragments does not hinder cloning. Following the In-Fusion reaction, miniprep DNA was isolated from four colonies
and analyzed by NcoI + SalI restriction enzyme digestion. All four minipreps were verified to contain all four pieces
of DNA (data not shown). Two clones were subsequently sequenced and were found to be error-free isolates of the 1.5 kb
coding region containing the PD-L2-IgG2a Fc-IgA tailpiece fusion (Table II). We then linearized the plasmid by MluI
digestion, transfected the construct into DHFR-deficient CHO cells, and selected for DHFR expression. A production
run yielded 12 mg/liter of purified 65 kDa fusion protein (Figure 2). This data shows that the In-Fusion technology
can be used to seamlessly join three or more pieces of DNA to create a multimeric, highly expressed, soluble
recombinant antibody.
Discussion
We observed fewer background colonies when the insertion site in a plasmid vector was created by digestion with two
restriction enzymes rather than one. Treating the vector with phosphatase was unnecessary and reduced cloning
efficiency. Accurate quantification and ratios of vector and DNA segments were the most critical factors for successful
multipiece In-Fusion cloning. Of the17 constructs generated, 28 out of 38 sequenced clones were found to be errorfree, and none required sequencing of more than 3 isolates to obtain the desired sequence. The most common error
found in incorrect isolates was a 1 or 2 bp deletion in a junction region, which could have resulted from an error
in the repair reaction due to incomplete conversion of the 15 bp overlap region to a single-stranded form. The use
of highly competent E. coli (1 × 109 cfu/µg) is recommended for multipiece In-Fusion reactions because the number
of transformants decreases as the number of pieces of DNA in the In-Fusion reaction increases. Two-, three-, and fourpiece In-Fusion reactions resulted in an average of 671, 539, and 56 colonies, respectively, on a plate spread with 0.1 ml
of a total 0.3 ml transformation reaction. In our experience, efficiency of the In-Fusion reaction does not depend
on the length of the DNA segments, which have varied from 83 bp to 12 kb with little difference in efficiency.
kDa
188–
98–
62–
49–
38–
1 2
Figure 2. Recombinant Antibody
Protein Production. One µg of purified
protein was analyzed using SDS-PAGE
under reducing conditions and stained
with Coomassie blue. Lane 1: Protein
molecular weight standards with size
in kDa indicated. Lane 2: Murine PD-L2IgG2a Fc-IgA tailpiece fusion protein.
Table II: Transformation Efficiency of a Four-Piece
In-Fusion Reaction
PD-L2
Extracellular
Domain
IgG2a
Fc
IgA
Tailpiece
Vector
693 bp,
6.1 ng
678 bp,
6.0 ng
100 bp,
0.9 ng
11,365 bp,
50 ng
Total
Transformants
No. ErrorFree/No.
Sequenced
120
2/2
28–
17–
14–
3
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Potential Applications
Multifragment cloning via In-Fusion facilitates the construction of modular vectors (Figure 3) as well as mutations
and synthetic genes (1). This can be accomplished by developing a toolbox of In-Fusion-Ready DNA modules that
can be readily joined in infinite combinations. Fragments can include fusion protein partners [Fc, green fluorescent
protein (AcGFP1), etc.], epitope tags, linkers, promoters, poly(A) sites, selectable markers, and origins of replication.
Adjacent modules can be designed to contain complementary 15 bp overhangs to allow restriction enzyme-independent
In-Fusion cloning of up to four modules simultaneously. If desired, unique restriction sites can be included in the primers
used to amplify the modules. Additional features can be added to the modular vectors by incorporating restriction
sites, translation initiation sites, linkers, or epitope tags in the primer sequences. Up to four pieces at a time may
be joined by In-Fusion reaction, creating a modular vector whose individual components may be readily excised
with restriction enzymes and/or replaced via In-Fusion.
This study shows that In-Fusion can be successfully used for multifragment cloning. The fact that multiple amplified PCR
products do not need to undergo any restriction digestion and can be used simultaneously for cloning into a linearized
backbone by a one-step reaction opens up new cloning possibilities. Two-piece In-Fusion reactions have already
been successfully used in high-throughput applications (4, 8). Adaptation of In-Fusion multifragment cloning
for high-throughput applications is the next step to further unleashing the potential of In-Fusion technology.
NcoI
Promoter 1
and splice
cDNA insertion site
SalI
Poly(A)-1
Modular
Vector
RS1
RS4
Figure 3. Modular vector. Red bars indicate junctions between segments. Different unique
restriction sites (RS) can be engineered into the junctions via the primers used to amplify
the segment. Each segment is amplified with primers that include 15 bp of overlap with
the adjacent segment. DNAs are joined via an In-Fusion reaction, so no restriction digest
is required. NcoI is a useful site because it includes a translation initiation site. MluI is a
convenient site for linearization of the vector prior to transfection.
Promoter 2
and splice
RS5
ori,
AmpR
RS6
MluI
Selectable
marker
Poly(A)-2
References
1. Zhu, B. et al. (2007) BioTechniques 43(3):354–359.
2. Evans, D.H. et al. (2003) In patent application 20030162265, USA,
pp. 1–52.
3. Hamilton, M.D. et al. (2007) Nucleic Acids Res. 35(1):143–151.
4. Marsischky, G. & LaBaer, J. (2004) Genome Res. 14(10B):2020–2028.
5.
6.
7. 8.
Latchman, Y. et al. (2001) Nature Immunol. 2(3):261–268.
Hirano, N. et al. (2006) Blood. 107(4):1528–1536.
Arthos, J. et al. (2002) J. Biol. Chem. 277(13):11456–11464.
Berrow, N.S. et al. (2007) Nucleic Acids Res. 35(6):e45.
Cat. #
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In-Fusion HD Cloning Plus
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