PRODUCT

PRODUCT

Large Circular DNA
Construction Tool

01TECHNOLOGY

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.

02APPLICATION

  • 01Artificially designed viral vector and plasmid construction
  • 02Amplification of long DNA and sequences difficult to amplify by PCR
  • 03Alternative to time-consuming and laborious E. coli cloning
  • 04Recombinant phage production
  • 05Efficient cloning of any DNA sequence, including cytotoxic and GC-rich sequences
  • 06The amplification products can be used directly for E. coli transformation, etc.

PRODUCT
OVERVIEW

OriCiro™ Assembly Kit

01REACTION

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.

02FEATURES

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.

03DISTINCTIVE
ADVANTAGES

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

01REACTION

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.

02FEATURES

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.

03DISTINCTIVE
ADVANTAGES

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)

PRODUCT
INFO

OriCiro™
Cell-Free Cloning System

01JAPAN

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.

OriCiro™ Assembly Kit
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
OriCiro™ Amp Kit
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.

02OTHER COUNTRIES

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.

OriCiro™ Assembly Kit
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
OriCiro™ Amp Kit
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.

PURCHASE

Profit customers

Non-Profit customers

OTHER COUNTRIES

OTHER

01FAQ

Assembly Reaction

Q1

Can I store the assembled products?

A1

Yes, you can store them at 4℃ for a few days. For further long-term storage, add final 5 mM EDTA before storage at -20℃. The following OriCiro Amp Kit tolerates up to final 0.5 mM EDTA.

Q2

Do I need purification of the assembly products before the OriCiro amplification?

A2

No. Add the assembly reaction (1 µl) directly into OriCiro Amp mixture (total 10 µl). OriCiro Amp selectively amplify circular DNA generated even slightly in the assembly reaction. The assembly intermediates (linear forms) are not amplified.

Q3

Can I use DNA fragments digested by restriction enzyme or CRISPR-Cas9?

A3

Yes.

Q4

No assembled products is observed.

A4

In certain conditions, the assembled product quantity may not be large enough to be observed after assembly reaction. It is highly recommended to perform following amplification reaction.

Amplification Reaction

Q5

Can I store the amplified products?

A5

Yes, you can store them at 4℃ for a few days. For further long-term storage, add final 20 mM EDTA before storage at -20℃.

Q6

How can I analyze the amplified products by electrophoresis?

A6

Dilute the 0.5 µl of products with 5 µl of Loading Buffer (25 mM Tris-HCl pH8.0, 25 mM EDTA, 0.1% SDS, 5% glycerol, 0.1% bromophenol blue), followed by agarose gel electrophoresis. The supercoiled products will be observed.

Q7

Extra bands are observed in agarose gel electrophoresis.

A7-1

DNA contamination.
Put negative control sample (reaction without DNA) for the amplification reaction. OriCiro Amp Kit amplifies DNA even from a single contamination molecule. To identify and remove DNA contamination, monoclonal amplification from a single DNA molecule following limiting dilution is useful.

A7-2

Concatemer DNA
When the amplified DNA size is less than 4 kbp, extra bands by concatemer DNA may appear. The situation may improve by shortening the reaction time.

Q8

No assembled products is observed.

A8-1

Please check the DNA size is less than 50 kb. If the target DNA size exceeds 50 kb, the amplification requires a different protocol than described in this manual. Please contact us at product@oriciro.com.

A8-2

The assembly reaction will be inhibited when salt and/or EDTA concentration of the DNA sample is too high.

A8-3

Increase the amplification reaction temperature up to 37℃.

A8-4

Increase the amplification reaction time up to 18 hours.

A8-5

Increase the template DNA amount added into the amplification reaction.

A8-6

Pre-incubate the amplification reaction mixture at 30℃ for 15 minutes just before adding the template DNA.

Q9

Can I reamplify the amplified products with OriCiro Amp Kit?

A9

Yes, Dilute the amplified products (~10-6), prepare new amplification reaction mixture and incubate again. We checked the amplified products can reamplify continuously more than 10 times.

Q10

Can I use a plasmid having a ColE1-type origin as a template?

A10

It cannot be used a plasmid only having a ColE1-type origin, the optimized oriC sequence is essential for OriCiro Amp Kit. If a plasmid has both oriC and ColE1-type origins, the amplification reaction is not affected by the ColE1 type origin.
02REFERENCES

"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]

03INTELLECTUAL PROPERTY
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)

04LABEL LICENSE

L0001 Limited Use Label License: For Research Use Only

The use of this product is limited to purchaser's own internal research. Purchaser cannot have any rights to use this product or its components for any purposes including but not limited to diagnostics, prophylactics, and/or therapeutics or otherwise clinical trials. No right to resell or transfer this product or any of its components is conveyed expressly, by implication, or by estoppel. With respect to any uses outside this label license, including, without limitation, (1) use in product manufacturing or quality control, (2) use in provision of services, information or data, and (3) resale this product, any of its components, any of its modifications, or derivatives made using this product, please contact OriCiro Genomics, Inc. by email at product@oriciro.com.

L0011 Limited Use Label License: Methods of Assembling and Amplifying Circular DNA (Not-for-Profit Entities)

This product (“Product”) and its use, is the subject of one or more issued and/or pending U.S. and foreign patent applications owned by OriCiro Genomics, Inc. End user (“Not-for-Profit End User”) of this Product should belong to a not-for-profit entity (i.e., academic organization, a university or another institution of higher education or any nonprofit scientific or educational organization including government agencies), provided, however, that when such Not-for-Profit End User uses this Products for other than its independent research use, such Not-for-Profit End User might need to obtain an appropriate patent license from OriCiro Genomics, Inc. Any and all Not-for-Profit End Users of this Product should submit a signed consent form, a copy of which can be found in this page, to OriCiro Genomics, Inc. or its authorized agent, prior to purchase, receipt and use of this Product. For license information, please contact OriCiro Genomics, Inc. by email at product@oriciro.com.

L0012 Methods of Assembling and Amplifying Circular DNA (For-Profit Entities)

This product (“Product”) and its use, is the subject of one or more issued and/or pending U.S. and foreign patent applications owned by OriCiro Genomics, Inc. End user (“For-Profit End User”) of this Product should obtain an appropriate patent license from OriCiro Genomics, Inc. even for its research use, prior to purchase, receipt and use of this Product. For license information, please contact OriCiro Genomics, Inc. by email at product@oriciro.com.
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