What is the biology of in vitro mRNA production from amplified DNA by PCR?
A little further clarification is needed; will the mRNA that you're asking about be translated? Do you need the 5' end capped and a poly A tail added? Two products I've used in the past are Megascript and MessageMachine and both manuals have very useful information. https://www.thermofisher.com/order/catalog/product/AM1334?SID=srch-srp-AM1334 and https://assets.thermofisher.com/TFS-Assets/LSG/manuals/1345M.pdf and
Yes, and I need 5' capping along with poly A tail added. Jeffrey Shelton
Thanks for clarification. You can certainly buy everything you need separately but this is a time I'd recommend buying a kit with all the components needed for capping and A-tailing in an optimized package with a control.
You'll need to incorporate a T7 RNA polymerase promoter sequence into one of your PCR primers. How you design your primers is very important in whether you transcribe sense strand or antisense. Message machine kit has T7 RNA polymerase, the 4 rNTPs, the cap analog and also includes a polyA polymerase. Take a look at the message machine manual and it will be very helpful.
Thank you so much Jeffrey Shelton Do I still use T7 RNA polymerase promoter sequence when I don't need 5' end capping or poly A tail?
Hi Sumit. I"m on holiday visiting family with limited internet resources so not able to draw an example but this weekend I can draw 3 different approaches I have taken in the past if you wish. You said PCR to IVT and you last post you said you don't need 5' cap or the poly A tail. This is a long reply, my apologies but I'm trying to cover each of these in some detail. If you take the enzymatic synthesis, then yes you will need a RNA polymerase promoter sequence directly upstream of your region of interest even if you don't require 5' capping or 3' A tailing.
3 easy approaches are:
1) gBlock from Integrated DNA Technologies. IDT can make dsDNA of your chosen sequence but you are limited on the size. You would need to add a RNA polymerase promoter sequence (commonly used RNA polys are T7, T3, SP6. There are others but these are most common in my opinion) Size and cost in $USD were last year $89.00 up to 500 bp, up to 1000 bp = $149, 2000 bp = $329, I don't remember the 3000 bp. you can perform IVT directly from the gBlock you receive. Design the gBlock with PCR primers at the 5' and 3' end of gblock so you can amplify it in the future and have an unlimited supply.
2) PCR with your primers; add a RNA polymerase promoter to 1or both of the primers you use to ampligy your region of interest (ROI). You might need to optimize the annealing temperature a bit. I'd suggest including a T7 promoter on 1 primer and an SP6 promoter on your other PCR primer. PCR primer annealing temps will need to be tested but a good place to start is your current PCR primer temp for 2 or 3 cycles followed by raising the temp 5 to 7 C for 25 to 28 cycles. Always check your PCR product on a gel to ascertain if a single amplicon or not. If single PCR product of expected size then go straight into IVT.
3) PCR amplify your region of interest with your current PCR primers. Clone into a plasmid vector which has a RNA polymerase promoter. Transform the plasmid into E.coli, and go through typical bacterial plasmid procedures. Once you isolate the plasmic make absolutely certain you linearize the plasmid with a restriction enzyme so you make only the ROI and not adjacent plasmic sequence.
If the RNA you need is short then you can have it synthesized by an oligo provider such as IDT or Dharmacon in the US.
If the RNA is longer than can be syntheized chemically then enzymatic synthesis is the next approach and will require that a RNA polymerase promoter be directly upstream of your ROI.
Hope this is helpful. I will have a bit of time these weekend to draw something but I'd recommend looking at the Megascript manual for a little more information.
The book I used for everything back in the 80's and 90s when I started was http://www.molecularcloning.com/ it is a great resource and is well worth the investment.
Thank you so much Jeffrey Shelton
I will have a look on the book you suggested.
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