126.96.36.199. Amplifying full-length viral RNAs
One critical factor in the successful amplification of viable, full-length cDNAs is the use of a thermostable DNA polymerase with proof-reading capacity. One such high-fidelity, high processivity DNA polymerase is Phusion DNA polymerase (New England Biolabs). If the fragment is to be cloned into a plasmid vector lacking a T7 or T3 RNA polymerase promoter site (required for the synthesis of full-length infectious RNA copies), then such a site should be incorporated into the 5’ amplification primer (e.g. Benjedou et al., 2002; Ongus et al., 2006).
1. Combine following in the 200 μl thin wall
PCR tube placed on ice:
1.1. 2 μl of the first strand cDNA reaction,
1.2. 35 μl sterile nuclease free water,
1.3. 10 μl of 5x HF Phusion amplification buffer,
1.4. 1 μl 10mM dNTP mixture,
1.5. 0.5 μl 2 μM “Reverse primer” (section 188.8.131.52.; step 3.2.)
1.6. 0.5 μl 2 μM T7 RNA polymerase promoter-tagged
“Forward primer” 5’-GCTATAATACGACTCACTATAGG(X)20-3’
where (X)20 are the first 20 nucleotides at the 5’ end of the virus genome
2. Mix well by pipetting on ice.
3. Add 2 μl (5U) Phusion DNA polymerase.
4. Mix by pipetting.
5. Place the tube in the thermocycler when the block is 90oC (Hot-start PCR).
6. Amplify with the following cycling programme:
6.1. 98°C:1 min,
6.2. 5x[98°C:15 sec – 52oC:60 sec – 72oC:7 min],
6.3. 25x[98°C:15 sec – 55oC:60 sec – 72oC:7 min],
6.4. 72oC: 7 min.
7. Purify the reaction products with PCR purification kit (Qiagen), eluting into 50 μl of water.