Fusion pcr and gene targeting in aspergillus nidulans

Berl R. Oakley, Edyta Szewczyk, Tania Nayak, Heather Edgerton, Yi Xiong, Naimeh Taheri-Talesh, Stephen A. Osmani

Research output: Contribution to journalArticlepeer-review

530 Scopus citations

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 (nkuAA) 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.

Original languageEnglish (US)
Pages (from-to)3111-3120
Number of pages10
JournalNature Protocols
Volume1
Issue number6
DOIs
StatePublished - Jan 2006

Bibliographical note

Funding Information:
ACKNOWLEDGMENTS This work was supported by grants GM31837 and GM042564 from the National Institutes of Health.

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