FIG. 4.

Localization of candidate mutants to individual wells of the Tn library. (A) First-round PCR search for mutants within the gene coding for gB using the UL55-rev primer. Each lane on the gel shows PCR products generated on secondary DNA pools representing all Tn mutants in one plate. A size marker (1-kbp ladder) is shown between lanes 4 and 5. (B) The PCR search was performed on the same DNA samples as in panel A but a primer specific for the gM gene (UL100-for) was used. (C) Pooled DNA samples representing the mutant clones stored in lanes A to H of plate 13 were subjected to PCR analysis with primers UL100-for, M13-for, and M13-rev. (D) All 12 individual wells (lanes 1 to 12) of row D identified in panel C were subjected to a third round of PCR screening. The desired mutant viral genome was detected in position D-11 on plate 13 of the Tn library. Pooled DNA from row D was included as a positive control (lane 13). Lane 14, PCR performed with the same DNA sample as in lane 13 using the distal primer UL100-rev with M13-for and M13-rev. The detection of the expected 1.2-kbp band was indicative of the absence of irregular deletion events.