Primer-dependent copying of rabbit globin mRNA with Qbeta replicase.

Abstract

THE synthesis of complementary strands of heterologous RNAs has been achieved by incubating these RNAs with Qβ replicase in the presence of Mn2+ (refs 1, 2). Qβ replicase has been used in this way for the in vitro copying of RNAs for which no replicating enzymes are available. A further extension of such synthesis is offered by the recently discovered primer-dependent RNA synthesis by Qβ replicase3. This approach is specially suited for the copying of eukaryotic RNAs (mRNAs) with a 3′-terminating poly(A) sequence. The complementary RNA synthesis is started at these poly(A) sequences with oligo(rU)6 as primer and continues into the heterologous part of the mRNAs. The particular advantages of this method are the independence of special structural features of the template RNA for the initiation of synthesis and the possibility to inhibit completely any disturbing 6S RNA synthesis which normally takes place in Qβ replicase incubations. Because of its defined start of synthesis, the newly synthesised complementary RNA may be very useful for sequence determinations of the template RNAs. I describe here experiments using rabbit globin mRNA as an example of the primer-dependent copying of eukaryotic mRNAs.