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. 2019 Aug 20;54(1):e89. doi: 10.1002/cpmc.89

Figure 3.

Figure 3

To verify the correct integration of the hygromycin B phosphotransferase (hph) marker in 45 different A. fumigatus putative gene knockout mutants, spore PCR was carried out with the primer pairs P1/hphsqR2 (A; amplification of the DNA spanning the upstream flanking region of the gene of interest and hph) and P4/hphsqF2 (B; amplification of the DNA spanning between the hph and the downstream flanking region of the gene of interest). Primers P1 and P4 are complementary to the upstream and downstream flanking regions of the gene of interest, respectively. Primers hphsqF2 and hphsqR2 target the sequence of the hph used to replace the genes of interest. The expected PCR band sizes for the correct knockout mutants are ∼1.5 kb. P: positive PCR control amplified from genomic DNA (50 ng) of a known A. fumigatus knockout strain; N: negative control (no DNA). (C) Spore PCR on the supernatant of the 45 A. fumigatus gene knockout mutants validated in Panels A and B using primer pair ITS1/D2. This PCR confirms the amplification of fungal rDNA and that the size of the correct amplification product is ∼1.2 kb. P: positive PCR control amplified from genomic DNA (50 ng) of the A. fumigatus wild‐type strain; N: negative control (no DNA).