By what methods do you synthesize long RNA ?
We synthesize long RNAs using two different methods.
1. The first method is chemical synthesis which allows us to prepare RNAs with the exact sequences. There is a length limitation up to 200 bases long and sometimes the yields can be lower because the yields for chemically-synthesized RNA decrease exponentially with increasing length. This is due to the coupling efficiencies at each step which are typically 90-99% (depending on the type of incorporated base). However, chemical methods allow the incorporation of flexible modifications at specific sites. Usually there are no issues with secondary structures during synthesis. However, they may be encountered during the purification stage.
2. The second method for generating long RNA is by using bacteriophage T7 polymerase or SP6 RNA polymerase. This enzymatic in-vitro-translational (IVT) method allows us to make extremely long RNA oligonucleotides. However, sometimes this technique generates three extra bases at the promoter sequences. Luckily, for most molecular biology applications, these 3 extra bases won't have a noticeable affect.
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10/28/2013