A method for single restriction enzyme-based scarless DNA cloning

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DNA assembly is one of the most important technologies for synthetic biology and is the process of physically linking together multiple fragments of DNA. By linking shorter DNA fragments end-to-end into longer sequences, longer assemblies of DNA fragments can be constructed before being joined to a vector for cloning.

Traditionally, DNA assembly has been limited to short lengths of DNA that can be assembled, and the process may involve many complex steps involving multiple enzymes, which can be very time-consuming and costly.

Academics from the University of Oxford have invented a new method for hierarchical DNA assembly, which provides a number of advantages over traditional methods. It can assemble fragments up to 200kb long and uses a single restriction enzyme throughout the process.  It also produces “scarless” assemblies, whereby the joins between fragments have no unwanted artefacts left over from the DNA assembly process.

DNA cloning is the cornerstone of synthetic biology and is the process of physically linking together multiple smaller DNA fragments into a large fragment.

Restrictions of traditional systems

Traditional DNA cloning systems using type IIS restriction enzymes require two or more enzymes for hierarchical assembly of large DNA constructs. This process is particularly complex and expensive because the DNA fragment to be cloned need to be free of the restriction sites for the enzymes used. The existing type IIS restriction enzyme-based DNA assembly systems also leave unwanted “scar” sequences in the assembled DNA. There is a widespread need for a DNA cloning method that addresses these limitations and improves the ability to assemble larger sequences of DNA.

A single restriction enzyme system

Oxford University academics have developed a new method for hierarchical cloning of DNA fragments, which is less complex than traditional methods whilst producing longer assembled fragments, potentially without “scar” sequences.

Other advantages of the Oxford method include:

  • The use of a single restriction enzyme throughout different stages of DNA cloning, which reduces the number of restriction sites to be avoided during the design stage
  • Can clone fragments over 200kb
  • More freedom to reuse cloned parts in modular DNA assembly
  • Option of hierarchical scarless cloning using a universal set of assembly vectors
  • Simple efficient replacement method for traditional cloning

Available for license

This technology is subject to a patent application and is available for license. Oxford University Innovation would like to hear from interested parties who may wish to commercialise this technology.

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