Cell-free protein synthesis using multiply-primed rolling circle amplification products

Utilizing in vitro transcription and translation (IVTT) to produce small quantities of proteins is convenient but requires a significant supply of pure template DNA. This can be cumbersome, particularly when the method is used for many different templates in a high-throughput manner. Multiply primed rolling circle amplification (RCA) with phi DNA polymerase is a simple way to generate large amounts of DNA; however, the products of this amplification method have interruptions in both strands and branched structures. In this study, we tested whether RCA-generated DNA can serve as the template for in vitro transcription. We found that RCA DNA-generated transcripts work in coupled in vitro translation with nearly the same efficiency (per nanogram of DNA) as those obtained from purified plasmid. We propose a convenient, single-tube format for template amplification, transcription, and translation.