In vitro method for the generation of protein libraries using PCR amplification of a single DNA molecule and coupled transcription/translation

A novel in vitro method for the generation of a protein library has been developed using the polymerase chain reaction (PCR) amplification of a single DNA molecule followed by in vitro coupled transcription/translation. DNA template encoding green fluorescent protein of a jellyfish Aequorea victoria was extensively diluted to one molecule per well, and then amplified by a total of 80 cycles of PCR with nested primers. The exact number of origins in the amplified DNA fragment was then estimated by directly sequencing a part of the fragment, at which an individual template molecule was marked by PCR with a primer containing three randomized bases. Since the sequences obtained in 91 independent amplifications were diversified statistically, each amplified fragment was likely originated from a single DNA molecule. In addition, the amplified fragments served as a template for in vitro coupled transcription/translation using T7 RNA polymerase and Escherichia coli S30 extract. These results suggest that the library obtained by the PCR amplification of a single DNA molecule diluted from a variety of DNA pools is potentially useful in high-throughput generation of protein libraries.