Gibson Deletion: a novel application of isothermal in vitro recombination

Swara Kalva,Paolo Mita,Jef D Boeke

doi:10.1186/s12575-018-0068-7

Swara Kalva, Paolo Mita + Show 1 more

Open Access

https://doi.org/10.1186/s12575-018-0068-7

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Abstract

BackgroundRecombinant DNA technology is today a fundamental tool for virtually all biological research fields. Among the many techniques available for the construction of a “custom DNA” molecule, the isothermal in vitro assembly, or Gibson assembly, allows for an efficient, one-step, scarless recombination-based assembly.ResultsHere, we apply and characterize the use of Gibson assembly for the deletion of DNA sequences around a DNA cut. This method, that we named “Gibson Deletion”, can be used to easily substitute or delete one or more restriction sites within a DNA molecule. We show that Gibson Deletion is a viable method to delete up to 100 nucleotides from the DNA ends of a cleavage site. In addition, we found that Gibson Deletion can be performed using single strand DNA with the same efficiency as using double strand DNA molecules.ConclusionsGibson Deletion is a novel, easy and convenient application of isothermal in vitro assembly, that performs with high efficiency and can be implemented for a broad range of applications.

Highlights

Recombinant DNA technology is today a fundamental tool for virtually all biological research fields
More recent approaches allow the assembly of DNA molecules without the use of restriction enzymes (PCR mediated assemblies [10, 11], Gateway system developed by Invitrogen) or ligase enzymes
The use of type IIS enzymes enabled the development of seamless cloning strategies able to assemble DNA molecules without the production of “inconvenient” scars left behind by the cloning process itself; the efficient Golden Gate cloning method is an example of these approaches [3]

Summary

Introduction

Recombinant DNA technology is today a fundamental tool for virtually all biological research fields. Recombinant DNA technology has given scientists the ability to edit, join, and delete DNA, driving fundamental discoveries in biology. This technology was initiated by the discovery of DNA ligase and restriction enzymes and it is still applied today for the construction of “custom made” DNA molecules. More recent approaches allow the assembly of DNA molecules without the use of restriction enzymes (PCR mediated assemblies [10, 11], Gateway system developed by Invitrogen) or ligase enzymes (ligation independent cloning or LIC [1]). Isothermal in vitro recombination, called “Gibson assembly” [4, 5, 7, 13], is a cloning method that assembles DNA molecules with overlapping sequences.

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