single-stranded regions with homology to anneal; a DNA polymerase, to fill in the gaps; and a DNA ligase, to seal the nicks between
the annealed and filled-in sequences.
SLIC (sequence and ligation–independent cloning) [17] and
In-Fusion Cloning (Clontech™, [16]) are based on the ligationfree cloning of overlapping PCR products treated by a DNA polymerase (the T4 and the vaccinia virus DNA polymerases, respectively), which possesses a 3
0 to 5
0 proofreading exonuclease activity.
The polymerase chews back one strand of each fragment, creating
single-stranded compatible ends. These overhangs are annealed
in vitro, and the recombinant circular construct is rescued in E. coli.
3.2 Primer Selection
for Homology-Based
Cloning
The assembly of a dual expression transfer plasmid involves the
combination of four DNA fragments with overlapping ends
which are in general obtained by PCR amplification: the vector
backbone, the dual promoter module, and the two cDNAs
(Fig. 3). Four pairs of oligonucleotides are required.
(a) The region of the plasmid including the divergent promoters
spanning from XhoI to BamHI sites is amplified with primers
named prom-F and prom-R, both including the restriction site
in their sequence and containing a Kozak consensus in their
5
0 end (see Table 2 for the primer sequences).
(b) The two genes are amplified by a PCR performed with the
primer pairs pH-gene-START-F/pH-gene-STOP-R and p10gene-START-F/p10-gene-STOP-R. They are composed of a
5
0 part corresponding to the cloning junction followed by 3
0
part complementary to the insert target (see step 4 below).
(c) Finally, the vector backbone, from XbaI to NheI sites, is
amplified with primers Backbone-F and Backbone-R. Note
that, alternatively to PCR amplification, the linearized vector
can be obtained by XbaI/NheI restriction digest followed by
gel purification (see Subheading 3.3).
Construct design and selection of primers for PCR amplification are detailed using an example where a dual expression cassette
for the co-expression of eGFP and mCherry is assembled (Fig. 4
and Table 2). We strongly advise using a DNA cloning software
(i.e., Serial cloner, ApE, SnapGene) to define the desired constructs, select a set of specific primers, and carefully simulate the
cloning procedure in silico before starting any experiment.
1. Identify plasmids/cDNAs from which the sequences encoding
the proteins of interest will be amplified and carefully verify
their nucleotide sequence. Do not hesitate to re-sequence if
cDNAs were obtained from external sources.
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
25
the annealed and filled-in sequences.
SLIC (sequence and ligation–independent cloning) [17] and
In-Fusion Cloning (Clontech™, [16]) are based on the ligationfree cloning of overlapping PCR products treated by a DNA polymerase (the T4 and the vaccinia virus DNA polymerases, respectively), which possesses a 3
0 to 5
0 proofreading exonuclease activity.
The polymerase chews back one strand of each fragment, creating
single-stranded compatible ends. These overhangs are annealed
in vitro, and the recombinant circular construct is rescued in E. coli.
3.2 Primer Selection
for Homology-Based
Cloning
The assembly of a dual expression transfer plasmid involves the
combination of four DNA fragments with overlapping ends
which are in general obtained by PCR amplification: the vector
backbone, the dual promoter module, and the two cDNAs
(Fig. 3). Four pairs of oligonucleotides are required.
(a) The region of the plasmid including the divergent promoters
spanning from XhoI to BamHI sites is amplified with primers
named prom-F and prom-R, both including the restriction site
in their sequence and containing a Kozak consensus in their
5
0 end (see Table 2 for the primer sequences).
(b) The two genes are amplified by a PCR performed with the
primer pairs pH-gene-START-F/pH-gene-STOP-R and p10gene-START-F/p10-gene-STOP-R. They are composed of a
5
0 part corresponding to the cloning junction followed by 3
0
part complementary to the insert target (see step 4 below).
(c) Finally, the vector backbone, from XbaI to NheI sites, is
amplified with primers Backbone-F and Backbone-R. Note
that, alternatively to PCR amplification, the linearized vector
can be obtained by XbaI/NheI restriction digest followed by
gel purification (see Subheading 3.3).
Construct design and selection of primers for PCR amplification are detailed using an example where a dual expression cassette
for the co-expression of eGFP and mCherry is assembled (Fig. 4
and Table 2). We strongly advise using a DNA cloning software
(i.e., Serial cloner, ApE, SnapGene) to define the desired constructs, select a set of specific primers, and carefully simulate the
cloning procedure in silico before starting any experiment.
1. Identify plasmids/cDNAs from which the sequences encoding
the proteins of interest will be amplified and carefully verify
their nucleotide sequence. Do not hesitate to re-sequence if
cDNAs were obtained from external sources.
Production of Multiprotein Complexes Using the Baculovirus Expression. . .
25