Here we propose a new general method for directly determining
RNA sequence based on the use of the RNA-dependent RNA polymerase
from bacteriophage φ6 and the chain terminators (RdRP
sequencing). The following properties of the polymerase render
it appropriate for this application: (1) the φ6 polymerase
can replicate a number of single-stranded RNA templates in vitro.
(2) In contrast to the primer-dependent DNA polymerases utilized
in the sequencing procedure by Sanger et al. (Proc Natl Acad Sci
USA, 1977, 74:5463–5467), it initiates nascent
strand synthesis without a primer, starting the polymerization on
the very 3′-terminus of the template. (3) The polymerase can
incorporate chain-terminating nucleotide analogs into the nascent
RNA chain to produce a set of base-specific termination products. Consequently, 3′ proximal or even complete sequence of many
target RNA molecules can be rapidly deduced without prior sequence
information. The new technique proved useful for sequencing
several synthetic ssRNA templates. Furthermore, using genomic
segments of the bluetongue virus we show that RdRP sequencing
can also be applied to naturally occurring dsRNA templates.
This suggests possible uses of the method in the RNA virus
research and diagnostics.