Rolling Circle Amplification for Sequencing Templates

Robust and reproducible isolation of high-quality templates is a requirement for successful DNA sequencing. To date, approaches for template generation have been limited to purification of biologically propagated M13 or plasmid-based templates, or in vitro amplification of such templates by polymerase chain reaction (PCR). In this chapter, we describe a protocol for a new approach to template generation: rolling circle amplification (RCA). We have found that templates produced through RCA yield more consistent and higher-quality sequence than identical templates generated from plasmid-prep methods. The protocol is simple, amenable to high throughput, and currently in use at the DOE Joint Genome Institute (Walnut Creek, CA) for the daily production of 30,000 sequencing templates.