Reverse transcriptase extra bases?

Bradley Turner bsturner at mbcrr.harvard.edu
Mon Aug 14 13:33:12 EST 2000


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.

Using the SMART(tm) cDNA system to map the transcription
initiation site
Biotechniques 28:846-852, 2000 May
B Gong, R Ge

Amplification of cDNA ends based on template-switching
effect and step-out PCR
Nucleic Acids Research 27(6):1558-1560, 1999
M Matz, D Shagin, E Bogdanova, O Britanova, S Lukyanov,
L Diatchenko, A Chenchik

CapSelect: a highly sensative method for 5' CAP-dependent
enrichment of full-length cDNA in PCR mediated analysis of RNA
Nucleic Acids Research 27(21):e31 (4 pages) 1999
WM Schmidt, MW Mueller

Generation and use of high-quality cDNA from small amounts of
total RNA by SMART(tm) PCR
A Chenchik, YY Zhu, L Diatchenko, R Li, J Hill, PD Siebert
Chapter 22, pp. 305-319 in Cene Cloning and Analysis by RT-PCR
eds. P Siebert and J Larrick, 1998 BioTechniques Books,
Natick, MA 

SMART(tm) RACE cDNA amplification kit User Manual
Clontech PT3269-1 March 23, 1999
<http://www.clontech.com/techinfo/manuals/PDF/PT3269-1.pdf>

SMART RACE to the finish: Clontech enters SMART RACE for cDNA
amplification
TheScientist 13(15):21, July19, 1999
D Swanson

SMART(tm) RACE cDNA amplification kit
CLONTECHniques pp.4-6 January 1999
<http://www.clontech.com/archive/JAN99UPD/smartrace.html>

Methods and compositions for full-length cDNA cloning using
a template-switching oligonucleotide
US Patent 5962272 Oct 5, 1999
A Chenchik, Y Zhu, L Diatchenko, P Siebert
Clontech Laboratories


Hope this helps,
Brad Turner [no affiliation with Clontech]
(Apparently SMART stands for "Switching Mechanism At 5'
end of RNA Transcript")

****************************************************************
                    Bradley Turner
                Beth Israel Deaconess
                    Medical Center

Harvard Medical School          617-667-1215 phone
Division of Gastroenterology    617-667-2767 fax
Room Dana 605                   bsturner at biosun.harvard.edu
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Boston, MA 02215                bsturner at mbcrr.harvard.edu
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On Mon, 14 Aug 2000, Eivind Hovig wrote:

> Is Superscript completely faithful at the end of a transcript, i.e. will 
> the enzyme add extra bases at the end? I experience 4 extra bases on a 
> PCR fragment of a size defined by a gene specific 3'transcriptase primer 
> and a 5' T7 polymerase sequence (as seen on the DNA level). As far as I 
> can find out, the RNA starts being transcribed from base 1 after the T7 
> polymerase recognition sequence. Thus, the DNA sequence is well defined, 
> and that leaves only the superscript to account for the extra bases, as 
> far as I can understand. Suggestions or pointers to obvious, but missed, 
> points are appreciated.
> 
> Sincerely
> Eivind Hovig
> 
> 
> 


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