Vector and 2-LTR circular DNA was detected using quantitative PCR assay based on a method previously described by Butler et al55 (link). Total cellular DNA was prepared using a Puregene DNA Purification Kit (Gentra Systems, Inc., Minneapolis, MN), from which a 250 ng or 500 ng sample was used for each reaction. To determine total, integrated, and 2-LTR circular vector DNA copy number, standard curves were generated by preparing serial dilutions of pcDNA-CS-CGW, genomic DNA from wild-type vector transduced HEK293 cells, or a 2-LTR circle reference plasmid that was generated upon the introduction of a 2-LTR fragment into a cloning vector, pCR4-TOPO (Invitrogen, Carlsbad, CA), respectively. Each sample was run in duplicate and data analysis was performed using Sequence Detection Systems 1.9.1 software (Applied Biosystems, Foster City, CA).
A 250 ng sample of total cellular DNA from transduced HEK293 cells at 3 days post-transduction was used for detection of 1-LTR circular vector DNA by standard PCR. 1-LTR circles were amplified using the following primers: 5’-GACCAATGACTTACAAGGCAGC-3’ (forward) and 5’-GCAAGCCGAGTCCTGCGTCG-3’ (reverse). The cycling conditions were 95°C for 10 min, thirty cycles of 92°C for 1 min, 56°C for 2 min, 72°C for 1.5 min, and 72°C for 7 min.
A 250 ng sample of total cellular DNA from transduced HEK293 cells at 3 days post-transduction was used for detection of 1-LTR circular vector DNA by standard PCR. 1-LTR circles were amplified using the following primers: 5’-GACCAATGACTTACAAGGCAGC-3’ (forward) and 5’-GCAAGCCGAGTCCTGCGTCG-3’ (reverse). The cycling conditions were 95°C for 10 min, thirty cycles of 92°C for 1 min, 56°C for 2 min, 72°C for 1.5 min, and 72°C for 7 min.