Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Analysis of Replicative Intermediates of Adeno-associated Virus through Hirt Extraction and Southern Blotting.

Bio-protocol (2017-07-01)

Martino Bardelli, Francisco Zarate-Perez, Leticia Agundez, Nelly Jolinon, R Michael Linden, Carlos R Escalante, Els Henckaerts

PMID28660237

ABSTRACT

Adeno-associated virus (AAV) is a small single-stranded DNA virus that requires the presence of a helper virus, such as adenovirus or herpes virus, to efficiently replicate its genome. AAV DNA is replicated by a rolling-hairpin mechanism (Ward, 2006), and during replication several DNA intermediates can be detected. This detailed protocol describes how to analyze the AAV DNA intermediates formed during AAV replication using a modified Hirt extract (Hirt, 1967) procedure and Southern blotting (Southern, 1975).

MATERIALS

Product Number

Brand

Product Description

77617

Sigma-Aldrich

Phenol – chloroform – isoamyl alcohol mixture, BioUltra, for molecular biology, 25:24:1

L3771

Sigma-Aldrich

Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)

W302600

Sigma-Aldrich

Sodium citrate dihydrate, ≥99%, FG

E9884

Sigma-Aldrich

Ethylenediaminetetraacetic acid, ACS reagent, 99.4-100.6%, powder

S3264

Sigma-Aldrich

Sodium phosphate dibasic, for molecular biology, ≥98.5% (titration)