Here we propose a new general method for directly determining RNA sequence based 
on the use of the RNA-dependent RNA polymerase from bacteriophage phi6 and the 
chain terminators (RdRP sequencing). The following properties of the polymerase 
render it appropriate for this application: (1) the phi6 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.