Lawrence Berkeley National Laboratory

The generation of DNA sequencing templates is typically inconsistent and labor-intensive procedure, especially in a high-throughput facility. Purification of recombinant plasmids from E. coli with subsequent PCR amplification of corresponding inserts has generally been employed, but this requires many laborious/time-consuming steps and does not always yield suitable amount of high-quality templates to be used in downstream applications. Replication by rolling-circle mechanism is common among bacteriophages in nature. Recently, rolling-circle amplification (RCA) with Phi29 DNA polymerase has been applied in vitro to marker DNA sequences (using specific primers) and to circular cloning vectors (using random hexamer primers) to achieve their exponential amplification via the DNA strand displacement. The US DOE Joint Genome Institute has successfully implemented random-primed RCA into the high-throughput process for production of sequencing templates. Here, we describe the RCA-based plasmid amplification protocol, as well as several practical applications for thus amplified DNA in sequencing and related procedures.