title: Fusion PCR and gene targeting in Aspergillus nidulans.
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.
authors.affiliation: Department of Molecular Genetics, Ohio State University, 484 West 12th Avenue, Columbus, Ohio 43210, USA.
authors.forename: Edyta
authors.initials: E
authors.lastname: Szewczyk
authors.forename: Tania
authors.initials: T
authors.lastname: Nayak
authors.forename: C Elizabeth
authors.initials: CE
authors.lastname: Oakley
authors.forename: Heather
authors.initials: H
authors.lastname: Edgerton
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiong
authors.forename: Naimeh
authors.initials: N
authors.lastname: Taheri-Talesh
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Osmani
authors.forename: Berl R
authors.initials: BR
authors.lastname: Oakley
authors.forename: Berl
authors.initials: B
authors.lastname: Oakley
mesh_terms: D001233:Aspergillus nidulans / Q000235:genetics*; D018390:Gene Targeting / Q000379:methods*; D018014:Gene Transfer Techniques; D016133:Polymerase Chain Reaction / Q000379:methods*; D011523:Protoplasts