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Fusion PCR and gene targeting in Aspergillus nidulansEdyta Szewczyk1,C Elizabeth Oakley1,Yi Xiong1,AbstractWe describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. 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 (nkuA) 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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