3.6 | Insertion of a single host 287 bp host DNA to the AcMNPV genome detected by Southern blot analysis
It appears that the EcoRI-F and HindIII-I fragments are the only fragments that have shifted when EcoRI and HindIII fragments were compared between AcP3-P0 and AcP3-P10 (Fig. 5A and B; left panel). According to the AcMNPV genome sequence, the EcoR1-F and HindIII-I fragments contain the fp25k locus [7, 21]. To confirm these shifts were from the 287 bp host DNA insertion and that it only inserted once in the AcMNPV genome, the DNA on the nylon membrane was probed with the 287 bp host DNA by Southern blot analysis (Fig. 5A; right panel). The 287 bp DNA probe hybridized only to the EcoRI-F and HindIII-I fragments of the passaged AcMNPV. This indicates that the 287 bp host DNA inserted only once into the AcMNPV genome during Sf21 cell passage (Fig. 5A; right panel). Since REN fragments in Fig. 5A (left panel) did not show fragments smaller than about 2 kbp, we then confirmed this by a short run. We confirmed that the 287 bp host DNA was not in the short fragments in the short run of the agarose gel (Fig 5B)