Synthesis and amplification of microgram quantities of dsRNA from dsDNA template using Phi6 RNA Replicase

Phi6 RNA Replicase replicates denatured DNA strands using NTP as substrate to form double-stranded DNA-RNA hybrid molecules. Subsequently the Phi6 Replicase displaces the DNA strand from the hybrid duplex, creating a double-stranded RNA molecule. The displaced ssDNA molecule can again serve as a template molecule in the next amplification cycles.

Protocol:
1. Prepare the desired dsDNA template by PCR amplification. Use primers that contain 18-22 nt
template-specific sequence and an additional tail sequence at the 5’ end:

5’ GGAAAAAAA-N_(18-22) 3’

where N_(18-22) is the template-specific stretch in the primer.

2. Set up a dsRNA synthesis reaction using the following reaction conditions:
1x Phi6 RNA Replicase Buffer
20-100 ng/µl PCR-amplified template DNA
0.1-0.2 mM ATP, CTP, UTP
0.3-0.6 mM GTP

3. Denature template DNA by incubating the reaction mixture at 95°C for 2 min. Snap cool on ice.

4. Add 1 U of Phi6 RNA Replicase for a 40 µl reaction volume.

5. Incubate at 32°C for 1-4 h.

6. Purify the amplified dsRNA using standard methods if necessary for your downstream application.