A practical approach to generate suitable de novo synthesis RNA template for a flavivirus RNA-dependent RNA polymerase

Due to its high transcription efficiency, bacteriophage T7RNA polymerase （T7 RNAP） has long been utilized inbacterial and in vitro systems to generate large quantityof RNA for various purposes （Studier and Moffatt,1986）. However, heterogeneity at the 3＇-end of the RNAtranscript remains a major limitation for in vitro RNApreparation using T7 RNAP. When approaching the endof its DNA template, T7 RNAP tends to add a few extranucleotides not directed by the template strand sequence,resulting in a mixture of RNA products that are equal toor longer than the desired length （Milligan et al., 1987）.