Physical Mapping of Adeno-associated Virus Serotype 4 DNA

The organization of adeno-associated virus serotype 4 (AAV 4) DNA was probed by using restriction enzymes. The cleavage sites of the following enzymes were mapped: BglII, BamI, HincII, KpnI, PstI, SalI, SstI, and XhoI. The orientation of transcription on the physical cleavage map was determined by locating the fragments which contain the 3' end of the mRNA. Strand separation gels were run for HinII fragments of AAV 4 DNA. By hybridizing AAV 4 mRNA to the resolved strands, the polarity of the DNA strands was identified. Restriction digestion of AAV 4 DNA sometimes produced terminal fragments which migrated in agarose gels as doublets. However, when AAV 4 DNA was prepared devoid of any single-stranded nicks, these terminal doublet bands were not observed upon subsequent restriction analysis. During these studies, the molecular weights of both AAV 4 and AAV 2 duplex DNA were measured and were found to be somewhat larger than previously reported (3.18 x 10(6) and 3.10 x 10(6), respectively).