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 (nkuA|[Delta]|) 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.NOTE:The version of this article initially published online contained the following errors: In the list of authors’ names, Berl Oakley should have been listed as Berl R Oakley. Figure 2: “Fusion PCR product”, “Chromosomal target” and “Recombined chromosome” labels were missing from each of the three panels, and an unnecessary “Upstream sequence” label appeared between panels b and c. Figure 3: “Histone H1” and “3' UTR” labels and corresponding bars were missing from panel a. p. 3113, under REAGENT SETUP: In the description of 1 M Tris–HCl solution, the last sentence should read “Store at room temperature (20 oC–27 oC) or at 4 oC." p. 3114, left column: In the third full paragraph, "tag4" should read "tag 4". p. 3314, Step 2: The last sentence should read: "We normally CsCl purify a large quantity of genomic DNA10 and use it for many experiments, but DNA purified in other ways will also work." The text above the table should read: "PCR reaction mix". The errors have been corrected in all versions of the article.