A capillary electrophoresis sequencing method for the identification of mutations in the inverted terminal repeats of adeno-associated virus.

ABSTRACT: Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed h

SUBMITTER: Mroske C 

PROVIDER: S-EPMC4015221 | biostudies-literature | 2012 Apr

REPOSITORIES: biostudies-literature