Non-Specificity for Primer Extension ?

[LIU at QUCDN.QueensU.CA]

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