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      Fusion PCR and gene targeting in Aspergillus nidulans.

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          Abstract

          We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.

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          Author and article information

          Journal
          Nat Protoc
          Nature protocols
          Springer Science and Business Media LLC
          1750-2799
          1750-2799
          2006
          : 1
          : 6
          Affiliations
          [1 ] Department of Molecular Genetics, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA.
          Article
          nprot.2006.405
          10.1038/nprot.2006.405
          17406574
          2ea43741-2838-47cb-8ca5-186922a06fca
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