PRODUCT
OriCiro™ Cell-Free Cloning System is an innovative tool enabling cell-free assembly and amplification of circular DNA molecules without E. coli transformation and culture. This product can not only streamline your workflow dramatically but also widen the scope of your genetic engineering techniques. This product is composed of OriCiro™ Assembly Kit and OriCiro™ Amp Kit and designed to produce the maximum effect when used in combination although each kit can be used independently.
In OriCiro™ Assembly Kit, DNA fragments are assembled via overlapping ends having homologous sequences. First, each end of the fragments is digested by exonucleases to expose a single stranded homologous sequence. Then, corresponding fragments having the homologous sequence each other are annealed by enzymes. The kit assembles up to 50 DNA fragments simultaneously in a single isothermal enzymatic reaction. The assembly products are subjected directly to OriCiro™ Amp Kit for selective amplification of your target circular DNA.
Efficient ligation
by isothermal
enzymatic reactions.
Up to 50 DNA fragments
can
be assembled together
as designed.
Up to
50 kb DNA fragment
can be assembled.
Ten fragments of 0.6 kb having 40 bp overlapping ends were assembled using OriCiro Assembly kit. The OriCiro kit (OriCiro) showed an assembly product of all ten fragments. DNA fragments before (-) or after (+) the assembly reaction were analyzed in agarose gel electrophoresis.
Assembly of 0.6 kb × 10 fragments
Total 6 kb,
Each fragments have 40 bp overlapping ends
Oriciro assembly | |
---|---|
Temperature | 42°C |
Time | 30 min |
OriCiro™ Amp Kit contains 26 kinds of purified enzymes that are essential for
the E. coli chromosome replication cycle. This Kit enables to amplify exponentially circular DNA having oriC (E. coli chromosomal origin)
in an isothermal condition even from a single template molecule. Up to 1 μg circular DNA can be yielded as supercoiled form per 10 μl reaction.
For more detailed explanation,
refer to the relevant publication.
in vitro reconstruction
of the E. coli genome
replication system.
More than 1 billion times
amplification from 1 circular
DNA molecule by isothermal
reaction for several hours.
Amplification of large circular
DNA exceeding 1 Mbp.
※Our products are optimized for up to
50 kb.
Up to 10,000 times
more accurate amplification
than PCR.
Consecutive amplification
for many generation (passage)
is possible by subculturing
in a new reaction solution.
Amplification product
can be transformed
into E. coli without
purification step.
Sequences with GC-rich which are difficult to amplify by PCR or
E. coli can be amplified.
Please refer to the table below for the advantages of OriCiro amplification technology over the conventional methods of E. coli cloning and PCR.
PCR | E. coli cloning | OriCiro Technology | |
---|---|---|---|
DNA size | <10kb | <50kb | <50kb |
Operation | Thermal cycling instruments | Transformation, Culturing and Purification | Very simple process Several hours of isothermal incubation |
Biosafety | Cell-free | Recombinant DNA experiment | Cell-free |
Fidelity |
+ (10-4~10-6 error/base/cycle) |
+++ (10-10 error/base/generation) |
++ (10-8 error/base/cycle) ※1 |
Sequence applicability | Not applicable to GC rich and repeat sequences | Not applicable to sequences that cause cell toxicity | Applicable to any sequence |
Product | Linear DNA | Circular DNA | Circular DNA |
※1 Technology under development to mach E. coli cloning.
COMPARISON TABLE (pdf)
Component
Catalog
Size
Price
OriCiro™ Assembly Kit
OriCiro™ Amp Kit
MS0011-A
5+10 Reactions
¥50,000
※The prices above are for non-profit customers.
※If the target DNA size exceeds 50 kb, the amplification
requires a different protocol than described in this manual.
Component | 5 Reactions (5μl/reaction) |
Storage |
---|---|---|
2×RA Mix | 12.5 µl | below -70°C |
oriC cassette (50 pg/μl) | 5 µl | below -70°C |
Control fragment (1 ng/µl) | 5 µl | below -70°C |
Component | 10 Reactions (10μl/reaction) |
Storage |
---|---|---|
10X RE Mix | 10 µl | below -70°C |
5X Buffer I | 20 μl X 2 vials |
below -70°C |
5X Buffer II | 20 μl X 2 vials |
below -70°C |
※The prices above are for non-profit customers.
※If the target DNA size exceeds 50 kb, the amplification
requires a different protocol than described in this manual.
Component
Catalog
Size
Price
OriCiro™ Assembly Kit
OriCiro™ Amp Kit
MS0011-A
5+10 Reactions
$ 550
€ 460
£ 415
※The prices above are for non-profit customers.
※If the target DNA size exceeds 50 kb, the amplification
requires a different protocol than described in this manual.
Component | 5 Reactions (5μl/reaction) |
Storage |
---|---|---|
2×RA Mix | 12.5 µl | below -70°C |
oriC cassette (50 pg/μl) | 10 µl | below -70°C |
Control fragment (1 ng/µl) | 5 µl | below -70°C |
Component | 10 Reactions (10μl/reaction) |
Storage |
---|---|---|
10X RE Mix | 10 µl | below -70°C |
5X Buffer I | 40 μl | below -70°C |
5X Buffer II | 40 μl | below -70°C |
※The prices above are for non-profit customers.
※If the target DNA size exceeds 50 kb, the amplification
requires a different protocol than described in this manual.
Profit customers
Assembly Reaction
Q1
Can I store the assembled products?
A1
Q2
Do I need purification of the assembly products before the OriCiro amplification?
A2
Q3
Can I use DNA fragments digested by restriction enzyme or CRISPR-Cas9?
A3
Q4
No assembled products is observed.
A4
Amplification Reaction
Q5
Can I store the amplified products?
A5
Q6
How can I analyze the amplified products by electrophoresis?
A6
Q7
Extra bands are observed in agarose gel electrophoresis.
A7-1
A7-2
Q8
No assembled products is observed.
A8-1
A8-2
A8-3
A8-4
A8-5
A8-6
Q9
Can I reamplify the amplified products with OriCiro Amp Kit?
A9
Q10
Can I use a plasmid having a ColE1-type origin as a template?
A10
"Overcoming the Challenges of Megabase-Sized Plasmid Construction in Escherichia coli."Mukai, T., et al., ACS Synth. Biol., 9 (6), 1315 (2020). (doi: 10.1021/acssynbio.0c00008) [PMID:32459960]
"Efficient Arrangement of the Replication Fork Trap for In Vitro Propagation of Monomeric Circular DNA in the Chromosome-Replication Cycle Reaction."Hasebe, T., et al., Life (Basel), 8 (4), 43 (2018). [PMID:30257439]
"Exponential propagation of large circular DNA by reconstitution of a chromosome-replication cycle."Su'etsugu, M., et al., Nucleic Acids Res., 45 (20), 11525 (2017). [PMID:29036468]
APPLICATION / PUBLICATION No. | Expires | Countries | Status | |
---|---|---|---|---|
Amplification | WO2016080424 | 2035 | US, EP, JP, CN | US: Granted (10301672) JP: Granted (6262877) EP: Granted (3222717) CN: Under review |
WO2017199991 | 2037 | US, EP, JP, AU, BR, CA, CN,IL, IN, KR, RU, SG |
National phase | |
WO2018159669 | 2038 | US, EP, JP, AU, BR, CA, CN,IL, IN, KR, RU, SG |
National phase | |
Assembly | WO2019009361 | 2038 | WIPO | PCT application filed |
INTELLECTUAL PROPERTY(pdf)
L0001 Limited Use Label License: For Research Use Only
L0011 Limited Use Label License: Methods of Assembling and Amplifying Circular DNA (Not-for-Profit Entities)
L0012 Methods of Assembling and Amplifying Circular DNA (For-Profit Entities)
OriCiro provides reagent kits for assembly and amplification of large DNA up to 200 kbp for research in academia and public institutions.
OriCiro provides on-demand services to our partners based on our proprietary technologies including a service of creating custom-made libraries of large DNA molecules.
OriCiro offers a license for use of our cell-free assembly and amplification technologies for the purpose of the development and manufacturing of plasmid DNA and synthetic genes.
OriCiro provides reagent kits for assembly and amplification of large DNA up to 200 kbp for research in academia and public institutions.
OriCiro provides on-demand services to our partners based on our proprietary technologies including a service of creating custom-made libraries of large DNA molecules.
OriCiro offers a license for use of our cell-free assembly and amplification technologies for the purpose of the development and manufacturing of plasmid DNA and synthetic genes.