Reverse transcriptase extra bases?

Eivind Hovig ehovig at radium.uio.no
Mon Aug 14 13:45:11 EST 2000


In fact, I am actually using one of these techniques myself in another 
project... But then I started wondering about the fact that I have 
performed reverse transcription previously, but not noticed this 
phenomenon. This time, however, I depend on knowing the precise base 
number of the resulting sequence in my application. I have not sequenced 
it (and suspect it might be a hassle anyway, given the added bases 
likely terminal position). And also, I get a distinct band, not a smear 
(indicating that there is no variation in the number of bases added). So 
I still have the question of whether or not other people frequently 
experience this.

sincerely
Eivind Hovig




In article <Pine.GSO.3.96.1000814123555.26877A-100000 at mbcrr>, 
bsturner at mbcrr.harvard.edu (Bradley Turner) wrote:

> Hello Eivind,
> 
> I'm actually quite pleased to hear of your extra bases.
> That is, if the extra bases are located at the 5' end of your
> sequence and your starting mRNA contined its 7mGppp cap
> structure, and the extra bases are dG's.  Supposedly,
> some RT's (including SuperScript) have a tendency to add 
> extra non-templated bases (esp. dC's) to the extreme 
> 3'end of the newly synthesized cDNA.
> 
> Presumably, this is the basis of a number of new methods/
> kits to obtain the 5' ends of mRNA's, (see references
> below) which is what I'm interested in.
> 
[...]
> 
> 
> Hope this helps,
> Brad Turner [no affiliation with Clontech]
> (Apparently SMART stands for "Switching Mechanism At 5'
> end of RNA Transcript")
>






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