A Takara Bio Company
Steve Oh1, Gia Jokhadze, Tommy Duong, Magnolia Bostick, Andrew A. Farmer
Abstract
1 Corresponding Author
Clontech Laboratories, Inc., 1290 Terra Bella Ave., Mountain View, CA 94043
3
pU
Ac
30
TRP1
00
Accuracy
+
286
4
A
Junction 1
Three-Insert Multiple-Fragment Cloning
Vector + insert
Negative control (no insert)
Cloning accuracy
Results
635 colonies
401 colonies
1 colony
39 colonies
Junction 4
Junction 6
96% (25/26 correct colonies)
pU
C
n
igi
r
o
MBP
TRP
GGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATAT
ACGGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGA
89 colonies
111 colonies
392 colonies
1 colony
39 colonies
78 colonies
100% (26/26 correct colonies)
19% (5/26 colonies)
73% (19/26 correct colonies)
er
t4
Results
3500
in
TR
250 0
High cloning accuracy is obtained when cloning single and multiple inserts with In-Fusion Cloning. In-Fusion HD Cloning and Gibson’s
method were each used to clone single and multiple (3) inserts into puC19 (linearized with BamHI). Each cloning system was tested
under the recommended conditions of its own cloning protocol. In an effort to make a more direct comparison with In-Fusion
cloning, this multiple-insert experiment with Gibson’s enzyme mix was also run at the shorter In-Fusion reaction time. All cloning
reactions were then transformed into Stellar™ Competent Cells, and 1/10th of each reaction was plated. Colony counts and clone
sequence verification were used to evaluate the results of each reaction. The results of single-insert cloning were roughly
comparable between the two systems, but the In-Fusion Cloning technology distinguished itself with lower levels of background—
an asset seen to even greater extent in the multiple-insert experiment, where it was also found to have much higher cloning accuracy
than Gibson’s method.
s
5
insert
00
Cloning accuracy
TRP
5-insert_lacZα-MBP-TRP1
4583 bp
30
Negative control (no insert)
1,592
AAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGA
MBP
Vector + insert
Gibson’s Method
Incubate at 50°C for 15 min
Incubate at 50°C for 60 min
Results
++++
50
mo r
0
lac pro latceo
pelraacZα
to
inse r
rt 1
p
3. Gibson, D. G.,et al. (2009) Nature Methods 6(5):343–345
Results
263
LacZα
MBP
MBP
MBP
TRP
Vector
4500
Am
2. Lestini, R., et al. (2013) Nucleic Acids Research 41(22):10358–10370
Conditions
In-Fusion HD Cloning
Incubate at 50°C for 15 min
+++
AGAACCGTACTTCACCTGGCCGCTGATTGC
TCAACCTGCAAGAACCGTACTTCAC
2
ert
ins
1. Chen, C. G., et al. (2014) Nucleic Acids Research 42(4):e26
3-insert,
multi-fragment
C
Multiple Insert Cloning
For Research Use Only. Not for use in diagnostic or therapeutic procedures. Not for resale. Takara and the Takara logo
are trademarks of TAKARA HOLDINGS, Kyoto, Japan. Clontech®, the Clontech logo, In-Fusion, and Stellar are trademarks
of Clontech Laboratories, Inc. All other marks are the property of their respective owners. Certain trademarks may not
be registered in all jurisdictions. Clontech is a Takara Bio Company. ©2014 Clontech Laboratories, Inc.
MBP
B
40
0
100% (26/26 correct colonies)
—
Number of Colonies per Plate
0
100
References
Results
209
MBP
Single Insert Cloning
Conditions
• When cloning multiple fragments in a single In-Fusion reaction, increasing the overlap
between fragments from 15 bp to 20 bp improves cloning efficiency ~5- to 6-fold.
—
ATCTGGATTAACGGCG
TCTGGATTAACGGCGATAAAGGCTA
ATAAAGGCTA
AAGGTAAACTGGTAA
AAGGTAAACTGGTAATCTGGATTAACGGCG
Junction 5
Gibson’s Method
Incubate at 50°C for 15 min
242
GTGAGTAAGCTCACTCATTAGGCACCCCAGGCTTTACACT
Junction 3
In-Fusion HD Cloning
Incubate at 50°C for 15 min
Vector only
Vector
Junction 2
0
2
• In-Fusion Cloning is more accurate than Gibson’s method when multiple inserts
are cloned simultaneously.
1/20
Five-Insert Multiple-Fragment Cloning
Increasing the length of overlaps from 15 bp to 20 bp improves the efficiency of multiple-fragment cloning.
Conclusions
8.5/10
91.5%
Fast, easy cloning without ligation. Panel A. In-Fusion® Cloning is a complete, optimized system that allows ligationindependent, directional cloning of PCR products into any vector, at any site of linearization in just one step. Your linearized vector
is combined with your PCR-amplified insert and the In-Fusion enzyme mix. The cloning reaction takes as little as 15 minutes and is
immediately ready for transformation into competent cells without further treatment. This powerful cloning technology is seamless,
accurate, and adaptable to a wide range of applications, including multiple-fragment cloning, large-fragment cloning, site-directed
mutagenesis, the addition of small tags to your gene of interest, direct cloning into large expression vectors, and high-throughput
experiments. Panel B. The In-Fusion Cloning system has been successfully used by many high-throughput users worldwide.
This bar graph shows the percentage of correct clones obtained during generation of an ORF collection by the Harvard Institute
of Proteomics. 3,367 individual clones were generated in order to obtain the desired 2,000 ORFs, with cloned inserts ranging
in size from 500–3,500 bp. Full-length sequencing of the inserts determined that over 3,000 of these clones contained the correct
insert, delivering over 91% cloning accuracy.
In-Fusion reaction
1/20
15-bp overlap
(blue/white)
20-bp overlap
(blue/white)
Dilution
1/10
1/10
Vector only
0/5
0/5
5-insert,
multi-fragment
21/5
115/12
0
Incorrect insert
Mutations
3,081
15 0
Correct insert
8.5/10
Increasing the length of fragment overlaps from 15 bp to 20 bp improves cloning efficiency for three-insert multiple-fragment cloning.
Panel A. 1.1 kb, 700 bp, and 600 bp inserts (20 ng each) were cloned into a 2.5 kb linearized vector (100 ng). 1/20th, 1/10th and the remainder
of the cloning reactions were plated. Panel B. Parallel experiments were performed with either 15-bp or 20-bp fragment overlaps.
15 bp was determined to be the optimal length for overlaps using the original In-Fusion Cloning kit. We tested whether the optimal
overlap length differed for the current In-Fusion HD enzyme and if this could improve the efficiency of multiple-fragment cloning.
Using 20-bp overlaps improved efficiency by ~6-fold. Plasmids were purified and sequenced. 100% of readable sequences were correct.
Number of Clones
Deletions
20-bp overlap
+++/++++ represent the relative estimation of colonies on plates with too many colonies to count.
00
t3
Multiple
fragments
20
insert 3
15-bp overlap
250 0
Overall Study Results
Small
tags
P
se
0
Length of cloned DNA fragments
HTP cloning in collaboration with researchers at Harvard
Single
fragments
15 0 0
rt 2
20
Number of Colonies per Plate
Dilution
3-insert_MBP-AcGFP-TRP1
4871 bp
ins e r
Construct
or
400
0
40
1–2
0
While In-Fusion Cloning simply and accurately clones single inserts into any vector
of choice, cloning applications are becoming increasingly more complex. In traditional
cloning systems, increasing the number of fragments can have a detrimental effect
on both efficiency and accuracy. However, In-Fusion Cloning maintains the high
accuracy typical of single-insert cloning even under more challenging demands,
by taking advantage of the host organisms’ highly evolved repair machinery. Here,
we improved cloning efficiency of multiple-insert cloning by extending the overlapping
sequence (see image below), and increasing the stability between fragments.
60
C
Amp
15 bp overlap
between insert
and vector
80
350 0
Introduction
Any
vector
lac prom
ote
r
00
45
n
igi
in
PCR
100
0
100
Any
insert
B
0
501
–75
0
751
–1,
00
0
1,0
01–
1,2
50
1,2
51–
1,5
00
1,5
01–
1,7
50
1,7
51–
2,0
00
2,0
01–
2,2
50
2,2
51–
2,5
00
2,5
01–
2,7
50
2,7
51–
3,0
00
3,0
01–
3,2
50
3,2
51–
3,5
00
Complete System
A
GF
B
0
A
Overlaps were Increased from 15 bp to 20 bp
for Three-Insert Multiple-Fragment Cloning
201
–50
1
In-Fusion Cloning: Seamless,
Ligation-Independent Cloning
rt 1
se
in
BP
M
In-Fusion Cloning technology enables directional, seamless cloning of any PCRamplified insert into any linearized vector in a single 15-minute reaction. No additional
treatment of the PCR fragment is required (such as restriction digestion, ligation,
phosphorylation, or blunt-end polishing). Instead, the In-Fusion enzyme generates
short regions of single-stranded overlaps between vector and insert(s), facilitating
accurate, directional cloning of the desired fragments. This overlap is designed
into the PCR primers used to amplify the desired insert sequences. While cloning
single inserts is an extremely efficient process, cloning experiments are becoming
increasingly challenging. Multiple inserts must be cloned, and kept in frame, in order
to build complicated constructs, to stitch genes together from synthetic building
blocks, or to engineer new functionalities through combinatorial protein domain
swapping, etc. Here, we present data showing how multiple-fragment cloning
efficiency and accuracy can be significantly increased by optimizing various
parameters of the In-Fusion Cloning reaction. This further extends the versatility
of the In-Fusion Cloning system to meet the coming challenges of innovative research
in the synthetic biology field.
Percentage of clones with the correct
full-length insert sequence
24
Optimized In-Fusion Cloning System Demonstrates High Efficiency
and Accuracy of Multi-Fragment Cloning
Clontech Laboratories, Inc.
P1
20
00
Increasing the length of overlaps from 15 bp to 20 bp improves cloning efficiency for five-insert multiple-fragment cloning.
Panel A. Five dsDNA inserts were designed to contain 20-bp overlaps between fragments. A comparison to 15 bp overlaps was tested
by shortening the ends of insert 2 by 5 bp (highlighted in yellow). Panel B. 450–480 bp inserts (20 ng each) were cloned into a 2.5 kb
linearized vector (33 ng). 1/10th of the cloning reactions were plated. lacZα (insert 1), which turns cells blue in our system, was included
for rapid identification of positive clones. Panel C. Shortening the ends of just one insert decreased the cloning efficiency by ~5-fold.
Blue colonies resulting from the 20-bp overlap reactions were further analyzed by colony PCR, and 10/10 were found to contain
a band corresponding to the five-insert product (data not shown).
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