FIG. 4.

Southern blot analysis of UL27 deletion mutants. Viral genomic DNA extracts were digested with enzyme PstI, electrophoresed, and blotted to nylon. Multiple replicates of the three extracts (AD169, T2092, and T2182) were run on the same gel and blotted to the same membrane and then cut into strips and hybridized separately with one of five different probes representing the sequences of the UL26 to UL29 genes and GFP. For wild-type AD169, two PstI genomic DNA fragments are detected, one with probes UL26, UL27, and UL28 and the other with probe UL29. T2092 and T2182, which have most of UL27 deleted, show an absence of hybridization with the UL27 (codons 1 to 415) probe. T2092 shows different fragment sizes with UL28 and UL26 probes because of two PstI sites in the GFP sequence inserted in place of UL27, whereas T2182 shows the same fragment with probes UL26 and UL28 because the GFP insert has been removed. The GFP probe hybridizes only to strain T2092 as expected, and neither strain T2092 nor T2182 shows the AD169 fragment size detected by probes UL26 to UL28, indicating absence of contamination with wild-type virus.