FIG. 5.
PCR evidence for pMAD10-mediated URA5Hc gene disruption in strain G184AS. Genomic DNAs from transformation recipient strain G184AS (lanes 1), the four hygromycin B-resistant, uracil-auxotrophic transformants (lanes 2 to 5), and a hygromycin B-resistant, uracil-prototrophic isolate (lanes 6) were subjected to PCR using primers targeted to a URA5Hc region upstream of the site of hph insertion (primer set A) or primers targeted to sites that flank the site of hph insertion (primer set B). The four uracil auxotrophs (lanes 2 to 5) have lost the native product and gained a new larger product due to hph insertion with primer set B. The uracil prototroph (lanes 6) shows both the unchanged native product and the larger product with primer set B, due to ectopic integration or linear plasmid formation by the transforming DNA. Lanes M, molecular size standards (λ/HindIII and φX/HaeIII).