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Advancing USER cloning into simpleUSER and nicking cloning.

Niels Bjørn Hansen1, Mette Lübeck1, Peter Stephensen Lübeck1

  • 1Section for Sustainable Biotechnology, Aalborg University, A.C. Meyers Vaenge 15, Copenhagen, SV DK-2450, Denmark.

Journal of Microbiological Methods
|November 5, 2013
PubMed
Summary

Two novel DNA cloning methods, simpleUSER and nicking cloning, streamline gene insertion into filamentous fungi vectors. These techniques simplify PCR fragment cloning, reducing time and complexity for genetic modification.

Keywords:
Co-transformationHigh-throughput cloningLigation-free cloningMultiple PCR fragment assemblySeamless DNA fusionUracil excision-based cloning

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Area of Science:

  • Molecular Biology
  • Mycology
  • Biotechnology

Background:

  • Traditional DNA cloning methods for filamentous fungi can be time-consuming and complex.
  • Efficient gene insertion is crucial for genetic modification of fungi like Aspergillus carbonarius.

Purpose of the Study:

  • To develop and evaluate novel, rapid DNA cloning methods for filamentous fungi.
  • To simplify the construction of vectors for gene insertion and co-transformation.

Main Methods:

  • Development of simpleUSER cloning, a modified USER cloning with a single enzymatic step.
  • Development of nicking cloning, which eliminates USER enzyme mix and PfuTurbo Cx polymerase.
  • Application of these methods for genetic modification of Aspergillus carbonarius.

Main Results:

  • Both simpleUSER cloning and nicking cloning effectively substitute USER cloning for single PCR fragment insertion.
  • The combination of simpleUSER and nicking cloning efficiently constructs selective plasmids and co-transformation plasmids.
  • Successful genetic modification of Aspergillus carbonarius using the developed methods.

Conclusions:

  • simpleUSER cloning and nicking cloning offer simplified, faster DNA cloning for filamentous fungi.
  • These methods reduce the time and complexity of vector construction for fungal genetic engineering.
  • The developed cloning strategies are applicable to various gene insertion and modification applications in fungi.