Reverse transcriptase extra bases?
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
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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