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Prune Dwarf Virus

Ed Rybicki ED at micro.uct.ac.za
Tue Jul 13 04:49:25 EST 1993

>     I am trying to clone PDV from single stranded RNA isolated from
> purified virus particles. I have no problem with the purification but
> am unable to clone it. I've been using MMLV RTase and random hexamers
> and cloning into Sma cut pGEM 4Z or Taq cutting the cDNA and cloning
> into a Acc I site (in pGEM 4Z). I'v also tried poly A tailing the
> RNA and priming with oligo dT's but with little success.
>     I would be very grateful if anyone could provide me with
> any suggestions regarding the cloning of any ilarviruses (or similar
> poly A minus RNA viruses). Thanks in advance for any help.

A thing to try might be going for dsRNA from plant, rather than ssRNA from
particles.  There are a number of good reasons for this:

1)  dsRNA is a LOT more stable than ssRNA, and isolation from plant tissue
    is EASIER than purification of virions (especially of something labile
    like an Ilarvirus or Cucumovirus), and subsequent purification of
    ssRNA from these, with attendant losses at all stages;

2)  you can poly-A tail dsRNA easier than ssRNA, and then you have TWO
    strands to prime with oligo-dT (it might be an idea to use
    3'-terminally degenerate oligo-dT; see Pappu et al., 1993, J Virol Meth

3)  there are a number of good protocols for cloning cDNA made from dsRNA
    by the rota- and reovirus people; basically, they rely on a single
    round of cDNA synthesis on two template strands, followed by NaOH
    treatment to get rid of RNA and annealing of the two complementary
    cDNAs, and Klenow fill-in reaction.  PCR is also a good option; you
    could do it on A-tailed dsRNA template with a single oligo-dT primer
    after cDNA synth in presence of DMSO.

Hope this was useful,

 | Ed Rybicki, PhD             |       "Lord, won't you buy me        |
 | (ed at micro.uct.ac.za)        |                                      |
 | Dept Microbiology           |         A Mer-ce-des Benz..."        |
 | University of Cape Town     |                                      |
 | Private Bag, Rondebosch     |                                      |
 | 7700, South Africa          |           - Janis Joplin             |
 | fax: 27-21-650 4023         |                                      |

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