Table 1.

Control experiments for rescue of circular intermediates in bacteria

Type of input DNASource of DNANo. of molecules (1010)No. of ampicillin resistant bacterial coloniesHead-to-tail circular intermediates present?e
Purified rAAVHirt DNA from infected muscle (22 day)3∼5 × 103 Yes
Virus added to uninfected muscle Hirt DNAa 30No
Linear ssDNA encompassing entire AAV genomeb Isolated from purified virus32No
Linear dsDNA encompassing entire rAAV genomeIsolated from proviral plasmid (HindIII/PvuII)c 33No
Linear dsDNA encompassing entire rAAV genome + ligased Isolated from proviral plasmid (HindIII/PvuII)3∼6 × 103 Yes
  • a Purified virus was added to muscle homogenates prior to preparation of Hirt DNA.

  • b Viral DNA predominantly contained single-stranded DNA (ssDNA) genomes, as evident by Southern blot analysis with the ITR probe (data not shown). However, there was also a small amount of double-stranded DNA (dsDNA) AAV genomes, likely due to reannealing of single-stranded genomes during preparation. Purified viral DNA concentrations were determined by optical density at 260 nm, and 75 ng, representing approximately 3 × 1010 viral genomes, was used for transformation of bacteria.

  • c HindIII/PvuII digestion was used to remove the entire rAAV genome from pCisAV.GFP3ori.HindIII and PvuII leave 10 and 0 bp of flanking sequence outside the 5′ and 3′ ITRs, respectively. The linear dsDNA fragment (4.7 kbp) was gel isolated following blunting with T4 DNA polymerase, and the DNA concentration was determined by optical density at 260 nm; 150 ng of linear fragment, representing approximately 3 × 1010 viral genomes, was used for transformation of bacteria.

  • d Linear dsDNA viral genomes (HindIII/PvuII-blunted fragment) were treated with T4 DNA ligase prior to transformation of bacteria.

  • e Confirmed by restriction enzyme (AseI, PstI, and SphI) digestion and Southern blotting against the ITR probe.