FIG. 2.

Kinetics of AAV site-specific integration. HeLa cells were infected with AAV-2 at an MOI of 500. Total genomic DNA was isolated at different times p.i. Purified DNA (1 μg) was preamplified in 50 μl for 13 cycles by conventional PCR. Samples of 2 μl were subjected to LightCycler PCR as outlined in Materials and Methods. (A) Raw data of the LightCycler analysis of HeLa cell DNA (1 μg) spiked with known copy numbers (10², 10³, 10⁴, and 10⁵) of standard plasmid pAAVS1-TR. (B) The copy numbers of AAV ITR/chr-19 integration site junctions per microgram of AAV-infected HeLa DNA were quantified by comparison to values of a standard curve run in parallel as outlined for panel A. Each value represents the mean ± the standard deviation of three independent cultures. (C) LightCycler PCR products were analyzed on agarose gels. Standards were 10¹, 10², 10³, 10⁴, and 10⁵ copies of pAAVS1-TR added to 1 μg of uninfected HeLa cell DNA. The bands in the range below 100 bp present in all lanes represent input primers. M, molecular size marker.