REPLICABLE RNA VECTORS - PROSPECTS FOR CELL-FREE GENE AMPLIFICATION, EXPRESSION, AND CLONING

This chapter discusses the possibility of utilizing the reproductive system of one of the positive-strand RNA viruses—Qβ bacteriophage, which infects Escherichia coli cells—for the amplification, expression, and cloning of foreign genes in vitro. This virus encodes a unique RNA replicase. In contrast to RNA replicases of other viruses, Qβ replicase can easily be isolated in preparative amounts and is long-lived under cell-free conditions. Qβ replicase is much more efficient in cell-free reactions than other systems utilized for the amplification of nucleic acids. It can produce as many as 1012 copies of RNA template in less than 30 minutes of incubation. The inability of Qβ replicase to amplify exponentially heterologous templates, including the genomic RNAs of related bacteriophages, has led to a wide-spread belief in the strict template specificity of this enzyme. At the same time, Qβ replicase does amplify exponentially numerous unrelated RNA species called “RQ RNAs.” © 1995 Academic Press Inc.