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Hum Gene Ther Methods. 2012 Feb;23(1):1-7. doi: 10.1089/hgtb.2011.104.

Systemic errors in quantitative polymerase chain reaction titration of self-complementary adeno-associated viral vectors and improved alternative methods.

Author information

1
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.

Abstract

Self-complementary AAV (scAAV) vector genomes contain a covalently closed hairpin derived from a mutated inverted terminal repeat that connects the two monomer single-stranded genomes into a head-to-head or tail-to-tail dimer. We found that during quantitative PCR (qPCR) this structure inhibits the amplification of proximal amplicons and causes the systemic underreporting of copy number by as much as 10-fold. We show that cleavage of scAAV vector genomes with restriction endonuclease to liberate amplicons from the covalently closed terminal hairpin restores quantitative amplification, and we implement this procedure in a simple, modified qPCR titration method for scAAV vectors. In addition, we developed and present an AAV genome titration procedure based on gel electrophoresis that requires minimal sample processing and has low interassay variability, and as such is well suited for the rigorous quality control demands of clinical vector production facilities.

PMID:
22428975
PMCID:
PMC3640491
DOI:
10.1089/hgtb.2011.104
[Indexed for MEDLINE]
Free PMC Article

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