Differential Display of Artefacts?

Stephen R. Lasky, Ph.D. Stephen_Lasky at brown.edu
Tue Dec 27 09:13:56 EST 1994


In article <3dnj1j$8cb at dns1.NMSU.Edu>, smori at nmsu.edu (Shahram Mori) wrote:


> I have been having problems too. I have been trying this in proc systems. I
> use random hexamers for the cDNA synthesis portion and 1 10mer for the PCR.
> A few times it has worked and many times it hasn't. I don't know why.
> Cheers,
> --
> Shahram Mori                                       _/\_
> Program in Molecular Biology                      _\  /_
> Dept. of chemistry and Biochemistry Box 3C        \_  _/
> NMSU  Las Cruces NM                                 ||
> 88003

Shahram:  I'm not quite sure how what you describe could work at all.  One
of the principles of the DD technique is the use of specific 3' primers
consisting of T12MN (where M is G,A orC  N is G,A,T, or C).  If you use
random primers for first strand synthesis, you have gotten rid of the
specificity of the T12xG (A,T, or C) reactions and have to rely on the
10mers to prime in both the 5' and 3' directions.  If I'm missing
something, please let me know.

One of the modifications that seemed to work when I was doing DD was to
use oligodT to prime the first strand synthesis and to use of the most
efficient cDNA synthesis kit I could find.  That was invitrogen's cycle
DNA kit when it had MeHgOH in it to denature the RNA.  I would run two
rounds of oligo-dT primed first strand synthesis and then use this with
the T12MN primers and the decamers in the PCR.  This seemed to give me
more reproducible results, but, according to Liang, may have caused the
under representation of some rare RNA's.  Despite this, I think that the
efficiency of first strand synthesis is key to the success of DD and I
will still use this modification when I get back to DD.

Hope this helps, but please let me know if I've missed something in your
description.

SRLasky

-- 
*********************************************************************
Stephen R. Lasky, Ph.D.
Roger Williams Medical Center/Brown University
Phone: 401-456-6572       Fax: 401-456-6569       e-mail: Stephen_Lasky at brown.edu
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"To me at least, 'Yuck' doesn't capture the full essence of death by
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