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 (nkuAΔ) 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.