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Updated: May 26, 2026

Targeted in Situ Mutagenesis of Histone Genes in Budding Yeast
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Targeted gene replacement in fungi using a split-marker approach.

Rubella S Goswami1

  • 1Department of Plant Pathology, North Dakota State University, Fargo, ND, USA. rubella.goswami@ndsu.edu

Methods in Molecular Biology (Clifton, N.J.)
|December 21, 2011
PubMed
Summary

The split-marker method enables efficient targeted gene replacement in filamentous fungi using PCR and homologous recombination. This technique facilitates functional gene characterization by replacing genes with a marker, simplifying genetic studies.

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Area of Science:

  • Molecular Biology
  • Mycology
  • Genetics

Background:

  • Targeted gene replacement is crucial for fungal gene function studies.
  • Genome sequencing advances facilitate gene-specific manipulation.
  • Filamentous fungi present unique challenges for genetic modification.

Purpose of the Study:

  • To describe and validate the split-marker method for targeted gene replacement in filamentous fungi.
  • To provide a streamlined protocol for fungal gene functional analysis.

Main Methods:

  • Utilizes PCR amplification of flanking regions and a marker gene.
  • Employs fusion PCR to create molecular cassettes.
  • Transformation of fungal protoplasts followed by selection and screening.

Main Results:

  • The split-marker method allows gene replacement without subcloning.
  • Efficient homologous recombination leads to successful gene knockout.
  • Transformants are identifiable through selective media and molecular screening.

Conclusions:

  • The split-marker method is a powerful and accessible tool for targeted gene replacement in filamentous fungi.
  • This approach simplifies functional genomics studies in fungi.
  • The protocol's efficiency and reduced steps make it suitable for widespread adoption.