April 15, 2009 - 10:23am
Allison Goldstein

Joined: Feb 26 2009
Can anyone recommend a good reverse transcriptase for performing genome sequencing on a RNA virus? I want to transcribe the RNA into 7kb long cDNA.
I also need a good enzyme for the long-range PCR, ideally amplifying 7kb. Reaction conditions (ideal temps, salt conditions, etc.) of the enzymes would be really helpful.
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I would use the AccuScript High-Fidelity Reverse Transcriptase for sequencing the RNA virus. It is a Moloney murine leukemia virus-based reverse transcriptase (MMLV-RT), however it possesses a proofreading 3’-5’ exonuclease activity and provides greater than three-fold accuracy compared to other available RTases that lack proofreading activity such as SuperScript III. Moreover, AccuScript High-Fidelity RT works well with templates up to 10 kb so it should work fine for your purpose. It further works at 37-42ºC which is good for minimizing RNA degradation.
To couple the high-fidelity of AccuScript RT, I would then use the KOD Hot Start DNA Polymerase for the long-range PCR. It provides extremely high fidelity and it has very high elongation rate and processivity, so you will have a much higher yield in correct product in shorter times. Moreover, using KOD Hot Start DNA Polymerase eliminates mispriming and primer-dimer formation, as well as primer degradation due to the presence of antibodies that inhibit the polymerase and exonuclease activities at room temperature. It works well up to 12 kb so it should be no problem for 7 kb, and extension is typically at 70°C (proofreading activity is optimal at 68°C whereas polymerization activity at 74°C so you can increase or decrease the extension temperature and adjust accordingly the extension time). Hope this helps!