FIG. 2.

Screening and verification of recombinant ORF57-null virus. A. DNA from bacterial colonies isolated by chloramphenicol and kanamycin selection was screened for the presence of sequences diagnostic of an insertion of the Kan^r cassette into ORF57 by PCR amplification, with one primer in ORF57 and one primer in Kan^r. Amplification of a 700-bp fragment is shown in DNA from three independent isolates (lanes 2 to 4), and molecular weight markers are shown in lane 1. B. Restriction analysis of recombinant BACmids was performed with HindIII digestion and electrophoresis. In addition to the presence of all expected fragments, recombinant BACmids contain a 3-kb HindIII fragment, as seen in lane 3 (arrow), instead of a 2-kb fragment seen in wild-type BACmid digests (*) (lanes 2 and 4), consistent with insertion of the Kan^r cassette in ORF57. C. Southern analysis of DNA from recombinant BACmid clones was used to verify correct insertion of Kan^r into ORF57. There is a HindIII site in the ORF57 gene which results in two fragments that hybridize to the ORF57 probe in wt KSHV (lanes 2 to 5). Homologous recombination and insertion of Kan^r into the smaller fragment result in two fragments approximately 3 kb in size (lane 1). Molecular weight standards from a HindIII digest of bacteriophage lambda DNA are shown in lane 1.