Quantitation of encapsidated recombinant adeno-associated virus DNA in crude cell lysates and tissue culture medium by quantitative, real-time PCR
JP Mayginnes, SE Reed, HG Berg, EM Staley… - Journal of virological …, 2006 - Elsevier
Recombinant AAV vectors are produced by transient transfection of mammalian cells. The
virus is usually purified from a combination of lysed cells and spent culture medium by
HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated
single-stranded viral DNA (ie DNA-containing virions) in cell lysates and the spent culture
medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative
DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility …
virus is usually purified from a combination of lysed cells and spent culture medium by
HPLC. We have developed a quantitative, real-time PCR assay for quantifying encapsidated
single-stranded viral DNA (ie DNA-containing virions) in cell lysates and the spent culture
medium. This requires extensive DNaseI digestion to reduce the amount of AAV replicative
DNA, as well as plasmid and cellular DNA, to negligible amounts. To demonstrate the utility …