differential display

RYBICKI, ED ED at micro.uct.ac.za
Mon Nov 1 23:27:07 EST 1993


> From:          cdolphin at uk.ac.crc (Dr. C. Dolphin)
> Subject:       differential display
....
> Specifically we are wondering whether the "anchored" oligo d(T)
> primers used in the initial reverse transcription really prime 
from the
> predicted 5' end of the polyA tail of the appropriate set of mRNAs 
in
> total RNA or are they annealing more non-specifically? it is
> possible that these "anchored" RT primers are annealing 
differentially
> BUT non-specifically.
> 
> Has anyone done this experiment? Does anyone using this technique
> have any comments?

If they are using simple oligo-dT primers, then you can bet they are 
annealing anywhere in the poly-A tail.  A simple mod to the primers 
is to synthesise (eg) a mix of 24-mer-dT+3'A,G,C (separately): this 
is GUARANTEED to anneal at the 5'end of the poly-A tail.

Used originally for sequencing cDNA clones primed using oligo-dT; 
since used for general pCR of poly-a+ potyviral RNAs.

Thweatt et al. 1990; Anal Biochem 190:314-316
Pappu et al. 1993; J Virol Methods 41:9-20

  ____________________________________________________________________
 | Ed Rybicki, PhD             |                                      |
 | (ed at micro.uct.ac.za)        |      "Lord, won't you buy me         |
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