A sequence-independent strategy for detection and cloning of circular DNA virus genomes by using multiply primed rolling-circle amplification.

ABSTRACT: The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with phi29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extract

SUBMITTER: Rector A 

PROVIDER: S-EPMC400362 | biostudies-literature | 2004 May

REPOSITORIES: biostudies-literature