Suitable templates for in vitro transcription - (Dec/06/2013 )
I'm using an in vitro transcription kit to prepare mRNA for cell transfection. The kit requires a linear DNA template with T7 promoter and 5' overhangs. I've e-mailed the manufacturer but I'm still awaiting a response. Does anyone know how long a sequence is needed before the T7 promoter and how much, if any, is needed after the stop codon?
Incidentally, for some reason the process is less efficient using blunt-ending enzymes and shouldn't be attempted using 3' overhangs. Does anyone know why that might be?
I can now answer all my own questions! Maybe this information will be useful to someone in the future:
Re: the length of sequence prior to the T7 promoter (i.e. 5'): 13 bp works, 23 bp works well (1).
Re: the length of sequence after the stop codon (i.e. 3'): no additional sequence is needed whatsoever (1).
Re: the in vitro transcription process shouldn't be attempted using a template with 3' overhangs: the transcripts deviate from the expected product (2). I don't have time to read the full article but just from the abstract it seems that when the template has 3' overhangs, the RNA polymerase doesn't detach from the template and a previously-synthesised transcript is then used as a template to elongate the sequence of the nascent transcript. The result is variable lengths of transcript with self-complementarity. Reducing the concentration of UTP in the reaction apparently fixes the issue but of course it's probably better to just use a template with 5' overhangs if possible (reducing UTP must limit the yield, right?).
1. Personal communication from Ronald Meis, Senior R&D Scientist, CELLSCRIPT.
2. Triana-Alonso, F.J. et al., 1995. Self-coded 3′-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase. Journal of Biological Chemistry, 270(11), pp.6298–6307.