Summary for "Non-specificity for Primer Extension?"

LIU at QUCDN.QueensU.CA LIU at QUCDN.QueensU.CA
Sun Apr 3 20:58:39 EST 1994


   About a month a ago I posted a request about the non-specific products
in my primer extension assay. One reply has been received so far. The
following is the repsonse from Martin Leech of Boston Univeristy. Thanks!
.bu.edu!user
Martin.(I was away from the net for a week. I apologize if I have missed
any other response. If I failed to acknowledge anyone, please let me
know. I shall repost my summary once I am informed of my mistake).

Various solutions to this.
Try adding actinomycin D, i believe this prevents extension products acting
as primers and causing non-specific extension.
Also verify the pe products using ribonuclease protection, s1
hybridization, or RT-pcr analyses.


.....          Martin Leach                Email:leach at mbcrr.harvard.edu
   _|____      Dept. of Pharmacology       Phone: (617) 638-5323
   / o  /      Boston Univ. School of Med. Fax:   (617) 638-4329
 _/  |-/__==/  80 E. Concord St. (L603)
(BULLDOZER) \_ Boston MA 02118            "Not the old underpants on your
               USA                           head.....WIBBLE" -BLACKADDER

Also attached is the original message I posted:
=========================================================================

Hello! Folks:

    I am trying to locate the 5' ends of viral DNA polymerase transcripts on
a virus genome by primer extension (PE) assay, but suffering from the
non-specificity of the assay system. I observed 2 PE products in both mock
and virus infected samples.

    The key reaction conditions used in my experiments are:
    1) RNA templates: 10-50 ug of total cellular RNA prepared from
       mock or virus infected cells.
    2) Primers: Primer A annealing to +15 to +24 relative to the
                         ORF (20-mer)
             or Primer B annealing to +67 to +97 relative to the
                         ORF (31-mer).
    3) Annealing temperature: 37-55 C
    4) PE temperature: 37-45 C

    Three possible causes for this non-specificity have been taken into
consideration:
    1) Sequence homology between the primer and host cell
       transcripts. But it is difficult for me to understand that
       why I) both primers (A and B) designed according to the
       viral sequence shares homology with cellular transcripts;
       and II) the distance between the primer and the 5' ends of
       the transcripts are the same for RNA of virus and cell
       origin.
    2) Mock sample contamination (with virus transcripts). But we
       have found aforementioned products in all 5 batches of RNA
       preparations. One of these RNA was prepared in the absence
       of virus infection.
    3) Reagent contamination. But it has been excluded by
       including a negative control composed of all reaction
       components except RNA template, which indeed did not
       produce any PE products.

    Therefore, I am urgently seeking advice from the net about:
    1) The possible explanation on the PE products in mock infected
       samples as well as the ways to get rid of those products (in mock).
    2) Alternative techniques to primer extension and nuclease
       protection assays to determine the 5'ends of the transcipts.

    Thank you for your attention!  I am looking forward to hearing from you.

                            Jian Liu
                            Dept. of Micro/Immunology
                            Queen's University
                            Kingston, Ontario, K7L 3N6
                            Canada
                            E-Mail: LIU at QUCDN.QUEENSU.CA



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