Multiply Primed Rolling-Circle Amplification Method for the Amplification of Circular DNA Viruses

INTRODUCTION

The use of whole genome amplification and analysis of viruses is of increasing importance, as data generated using these methods are currently used for clinical diagnostics, epidemiological studies, phylogenetic analyses, and studies of genome organization and evolution. The best known amplification method for DNA is the polymerase chain reaction (PCR). This technique, however, has drawbacks: PCR produces relatively small amplicons and also requires prior knowledge of sequence data for the construction of the required consensus or degenerate primers. For circular DNA templates, it is possible to overcome these drawbacks by using the multiply primed rolling-circle amplification (RCA) technique, which mimics the rolling-circle mechanism that occurs in nature for replication of circular DNA molecules, e.g., plasmids. The RCA protocol described here is optimized for the amplification of circular DNA virus genomes.