OriCiro Genomics

OriCiro Cell-free Technology



1.  DNA Assembly Technology

Hyper assembly of multiple DNA fragments

  • 1-step isothermal reaction, cell-free, easy-to-handle
  • Seamless assembly via overlapping ends (25 - 60 bp)
  • Simultaneous assembly of up to 50 fragments
  • Assembly of large DNA fregments up to 200 kb
         







   

2.  DNA Amplification Technology

In vitro reconstitution of chromosome replication cycle

  • Whole replication cycle of the E. coli chromosome is reconstituted in vitro using purified proteins.
  • The replication cycle repeats autonomously and continuously, allowing to "exponential" amplification of circular DNA.
  • Requirements for the template DNA are                      "circular configuration" and "oriC sequence (300 bp)."



Hyper amplification by chromosome replication system

  • 1-step isothermal reaction, cell-free, easy-to-handle
  • Up to 100 ng/µl supercoiled DNA is produced within 3 hr at 30ºC even from a single DNA molecule (10 kbp)
  • Extremely high fidelity
  • Applicable to wide-size range (1 kbp - 1 Mbp)
  • Applicable to any sequence (GC-rich DNA, repeat DNA, etc.)




3.  Synergetic effect by
Assembly & Amplification

Cell-free production of circular DNA
from multiple DNA fragments

  • Subject the assembly products directly to the amplification reaction.
  • Circular DNA (complete assembly product) can be amplified selectively from a single molecule level.
  • Unnecessary intermediate (linear DNA) is "shaken off"

Q&A

How much DNA can be obtained?
Typically 1.0 µg DNA / 10 µl reaction is obtained at 30ºC for 3 h.
Is it possible to re-amplify the RCR products? 
Yes. Products can be amplified successively in many times by diluting into the fresh amplification mixture and incubation.
How is the amplification fidelity? 
Because of the chromosome replication machinery, the fidelity is extremely high. The error rate is 1.0 × 10-8 errors / base / cycle which is 105 times lower than that of Taq polymerase (conventional PCR polymerase).
Are the assemby products or the amplification products able to use for E. coli transformation?
Yes. Use directly without any purification procedure. The oriC sequence acts as a low-copy plasmid origin in E. coli (no need to design an additional plasmid origin).