T7 RNA Polymerase

Chris Noren noren at neb.com
Thu Sep 22 12:44:36 EST 1994


In article <Pine.3.89.9409211456.A23425-0100000 at herman.cs.uoguelph.ca>,
ylee at uoguelph.ca (Yoon Lee) wrote:

> It seems that when I synthesize RNA in vitro with T7 RNA Pol and PCR-made 
> template, T7 puts one extra nucleotide at its 3' end. Obviously, the 
> addition of extra A by Taq polymerase during PCR is not the problem in 
> this case because T7 uses a DNA strand with exact 5' terminus defined by 
> oligomer as its template. Most likely explanation of this phenomenon may 
> be that T7 Pol itself is putting extra nucleotide during polymerization 
> process. If you are aware of this problem, please tell me how to prevent it?
> Thanks in advance.

The ability of T7 RNA polymerase to add non-template-encoded bases to the
3' ends of RNA transcripts has long been the bane for people making RNA for
physical/catalytic studies. Two literature descriptions of this phenomenon
are Milligan et al. NAR 15, 8783-8798 (1987) and Sampson & Uhlenbeck PNAS
85, 1033-1037 (1988). The identity of the extra added base apparently
varies by template: the first study showed a mixture of A and C, the second
showed G and C. The percentage of product with non-template-encoded
nucleotides ranges from 10-40%. If homogeneous product is desired, then
purification on long, high percentage polyacrylamide gels is the only
solution (short of engineering a self-cleaving ribozyme sequence at the 3'
end of the transcript!).

-- 
Chris Noren
New England Biolabs
(800)632-5227 ext 261
noren at neb.com

We Await Silent Trystero's Empire



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