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Szewczyk E, Nayak T, Oakley CE, Edgerton H, Xiong Y, Taheri-Talesh N, Osmani SA, Oakley BR (2006) Fusion PCR and gene targeting in Aspergillus nidulans. Nat Protoc 1(6):3111-20
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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.
Status:PublishedType:Journal Article|Research Support, N.I.H., ExtramuralPubMed ID:17406574

Topics addressed in this paper
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Topics
Genes linked to topics
nkuA
(A. nidulans)
Aspergillus nidulans/Emericella nidulans
Mutants/Phenotypes
Techniques and Reagents
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