Dr. Samuel Bru and his colleagues in Dr. Josep Clotet’s research group routinely purify recombinant chimeric proteins from S. cerevisiae; the group was looking for a fast, easy way to successfully clone the chimeric proteins their work requires. In this study, two methods were tested side-by-side for cloning single and multiple inserts: In-Fusion Cloning and ligation-based cloning. Cloning experiments using each method were set up for one, two, or three inserts, respectively. Colonies were screened by restriction digest, and cloning efficiency was determined as the number of positive clones obtained from ten randomly chosen colonies, averaged across five independent experiments.
By directly comparing the two cloning methods, they found that single-step multiple-fragment cloning with traditional T4 ligase was extremely difficult due to low cloning efficiency. However, when the experiments were performed with In-Fusion Cloning, the process was highly efficient for both single- and multiple-insert reactions, and was completed in a shorter time with less handling.
In conclusion, the In-Fusion HD Cloning Kit provided us with a higher cloning efficiency and faster results compared to traditional ligation based cloning for both single and multiple insert cloning."
—Dr. Samuel Bru
Results
For In-Fusion Cloning, inserts were designed with specific cloning ends that overlapped with adjacent DNA fragments (insert/vector or insert/insert; see Figure 1). For ligation cloning, inserts were designed with compatible restriction sites for adjacent fragments (insert/vector or insert/insert; see Figures 1 and 3). All inserts were PCR-amplified and purified, however, inserts for ligation cloning were digested with the appropriate restriction enzymes prior to purification.
Cloning reactions with one, two, or three inserts were set up for each cloning method (see Figure 1 and Table II), with the two- and three-insert reactions designed to clone all DNA fragments simultaneously into an expression vector. In all cases, the final plasmid was ~6.8 kb. Positive clones were identified by restriction digest, and the number of positive clones obtained from ten randomly chosen clones was averaged for five independent experiments to determine cloning efficiency (Figure 2). In-Fusion Cloning was successful in all tested conditions, providing high cloning efficiencies for both single and multiple inserts. Ligation cloning lagged behind, especially with the multiple-insert reactions.
Figure 1. Schematic representation of single- and multiple-fragment cloning reactions using In-Fusion Cloning and ligation-based cloning with T4 DNA ligase. Insert fragments are shown in linear form; vectors are shown in circular form. The 15-bp overlaps between adjacent DNA fragments (vector/insert or insert/insert) needed for In-Fusion cloning are shown in different colors. In the case of traditional ligation, the colors represent different cohesive ends.
Cloning reaction type: 1 insert
In-Fusion Cloning experiment
% Efficiency
1
100
2
90
3
90
4
100
5
100
Average
96
Ligation cloning experiment
% Efficiency
1
90
2
80
3
70
4
70
5
70
Average
76
Cloning reaction type: 2 inserts
In-Fusion Cloning experiment
% Efficiency
1
60
2
70
3
100
4
60
5
100
Average
78
Ligation cloning experiment
% Efficiency
1
0
2
10
3
0
4
0
5
0
Average
2
Cloning reaction type: 3 inserts
In-Fusion Cloning experiment
% Efficiency
1
30
2
50
3
60
4
50
5
20
Average
42
Ligation cloning experiment
% Efficiency
1
0
2
0
3
0
4
0
5
0
Average
0
Figure 2. Quantitative comparison of cloning efficiencies obtained with In-Fusion Cloning and ligation-based cloning methods for single- and multiple-fragment cloning reactions. Panels A. Bar graphs of average cloning efficiencies for all three cloning reaction types. Results correspond to the average of five independent experiments ±SEM. Asterisks represent significant differences calculated using the Mann-Whitney U test, with *** indicating a p-value <0.0005. Panels B. Cloning efficiencies are listed for each of the five individual experiments and as the average efficiency for each cloning reaction type.
Conclusions
In-Fusion technology outperformed traditional ligation-based cloning methods in direct comparisons of both single- and multiple-insert cloning experiments. While the ligation method had a cloning efficiency of 76% for single-insert cloning, efficiency fell significantly for multiple-insert cloning, with two- and three-insert reactions at 2% and 0% efficiency, respectively. In contrast, In-Fusion Cloning was 96% efficient for single-insert cloning, and also displayed good cloning efficiency with two- and three-insert cloning at 78% and 42% efficiency, respectively. Overall, In-Fusion technology was shown to be an easier, faster cloning method in terms of efficiency, number of steps, and handling time for all three reaction types.
Methods
In-Fusion Cloning insert fragments were amplified via PCR, followed by column purification with the Macherey-Nagel NucleoSpin Gel and PCR Clean-Up kit. Ligation inserts were also amplified via PCR, but were then digested with appropriate restriction enzymes for three hours (see Figure 3 and Table II) prior to column purification with the same clean-up kit.
Figure 3. Schematic of digests for multiple-insert cloning with ligase. Multiple-insert ligation cloning is possible if the linearized vector and inserts have compatible sticky ends, but only with their adjacent fragments. The top schematic shows the digests performed for the two-insert ligation reaction, while the bottom schematic shows the digests performed for the three-insert ligation reaction.
Linearization of the pGEX6P1 vector (~5 kb) was performed by restriction digest with BamHI and EcoRI for three hours, followed by affinity column purification with the Macherey-Nagel NucleoSpin Gel and PCR Clean-Up kit. This linearized vector was used as the backbone for all cloning reactions. For In-Fusion Cloning reactions, vectors and their respective insert(s) were incubated together with the In-Fusion HD Cloning Plus (Discontinued, replaced by Cat. #s 638945, 638946) enzyme mix for 15 min at 50°C. For ligation reactions, vectors and their respective insert(s) were incubated together with T4 DNA ligase for 4 hours at 20°C. (See Figure 1, above, and Table II, below for reaction diagrams and setups.) All cloning reactions were then transformed into competent E. coli cells.
Table I. Cloning protocols
In-Fusion Cloning protocol
T4 DNA ligase protocol
PCR amplify each insert fragment
Linearize pGEX6P1 vector (4984 bp) by restriction digest with BamHI/EcoRI enzymes (3 hr)
Purify vector and insert DNA using affinity columns
Incubate vector and insert DNA with the In-Fusion HD Cloning Plus enzyme mix (15 min at 50°C)
Transform In-Fusion Cloning reaction into competent E. coli cells
PCR amplify each insert fragment
Linearize pGEX6P1 vector (4,984 bp) by restriction digest with BamHI/EcoRI enzymes (3 hr)
Digest insert(s) with restriction enzymes (see Figure 3 and Table II, below) (3 hr)
Purify vector and insert DNA using affinity columns
Incubate vector and insert DNA with T4 DNA ligase (4 hr at 20°C)
Transform ligation reaction into competent E. coli cells
Ten clones were chosen at random for screening by restriction digest. Five independent experiments were performed for each reaction, and the number of positive clones (out of each ten screened) was averaged across the experiments in order to quantify cloning efficiency (Figure 2). Reaction setups are summarized in Table II below.
Table II. Reaction setups
Cloning Reaction 1 (1 insert)
Cloning Reaction 2 (2 inserts)
Cloning Reaction 3 (3 inserts)
In-Fusion Cloning
6 µl
Insert-1 (1,800 bp, 200 ng)
6 µl
Insert-2 (900 bp, 200 ng
6 µl
Insert-4 (600 bp, 200 ng)
2 µl
Vector linearized with BamHI/EcoRI (90 ng)
6 µl
Insert-3 (900 bp, 200 ng
6 µl
Insert-5 (600 bp, 200 ng)
2 µl
In-Fusion HD Cloning Plus enzyme mix
2 µl
Vector linearized with BamHI/EcoRI (90 ng)
6 µl
Insert-6 (600 bp, 200 ng)
10 µl
Total
4 µl
In-Fusion Cloning Plus enzyme mix
2 µl
Vector linearized with BamHI/EcoRI (90 ng)
2 µl
H2O
5 µl
In-Fusion Cloning Plus enzyme mix
20 µl
Total
25 µl
Cloning Reaction 1 (1 insert)
Cloning Reaction 2 (2 inserts)
Cloning Reaction 3 (3 inserts)
Ligation cloning
6 µl
Insert-1 digested with BamHI/EcoRI (1,800 bp, 200 ng)
6 µl
Insert-2 digested with BamHI/HindIII (900 bp, 200 ng)
6 µl
Insert-3 digested with BamHI/HindIII (600 bp, 200 ng)
2 µl
Vector linearized with BamHI/EcoRI (90 ng)
6 µl
Insert-3 digested with HindIII/EcoRI (900 bp, 200 ng)
6 µl
Insert-4 digested with HindIII/NotI (600 bp, 200 ng)
0.5 µl
ligase buffer
2 µl
Vector linearized with BamHI/EcoRI (90 ng)
6 µl
Insert-5 digested with NotI/EcoRI (600 bp, 200 ng)
