"LONG" RT/PCR

Terry Hanzlik terryh at ento.csiro.au
Wed Nov 23 17:53:07 EST 1994


Dear Steves and netters,
I also agree about the RT step limiting the length of the PCR
product.  My suggestion would be to use at least two RTs,
preferably BRL's Superscript II and anybody's AMV, in the same 
reaction.  We notice in RNA sequencing reactions that they pause
at different places and complement each other's ambiguous
sequence.  In addition, between adding additional aliquots of
RTs as suggested by Steve, incubate the reaction at 70-80 C for a 
few minutes to melt interfering secondary structures and quench
on ice.  Then use
one of the long range PCR stategies with longer than normal
primers to keep the annealing temperature high.  We have had luck
with both the Barnes and Perkin-Elmer procedures.  I wouldn't
bother with  RT'ing with Tth.

Using the above RT strategy, we obtained viral
cDNAs for greater 90% of the two genomic strands (2.5 and 5.3 kb)
of the tetravirus we work with.  Each of the strands look to have
horrific secondary structures with the smaller strand causing
M-MLV to predictably jump the same 50 bases in about 50% of the
products.  This was even seen on the RNA sequencing reactions as
well as the PCR products.

While on this subject, has anybody else had a similar exprerience
with a deletion artifact of reverse transcription?

Terry Hanzlik, terryh at ento.csiro.au
CSIRO Division of Entomology
Box 1700
Canberra, ACT  2601
Australia

> 
> In article <Pine.3.87.9411141647.B29380-0100000 at csuvax1> wylie at CSUVAX1.MURDOCH.EDU.AU (Steve Wylie) writes:
> >Has anyone tried RT/PCR for RNA of 10KB or longer? I have tried to do 
> >this with the potyvirus (BYMV) I work with, but so far no success. 4Kb 
> >isn't a problem but anything longer is! I suspect the RT to be the 
> >limiting step so have tried M-MLV and AMV RT's, incubating for 3-4 hours 
> >but still nothing. Any comments will be gratefully accepted.
> >Steve Wylie, Murdoch University, Western Australia 
> >
> I have managed 7.5 kb with alphavirus RNA and haven't (yet) tried longer PCRs.
> I would agree that the RT is probably the limiting step. It might be worthwhile
> to add the RT in multiple aliquots, say every 30-60 min over 2-3 hr. Maybe I'll
> give that a try. You might also try other enzymes, like Tth Pol (from ABI-
> Perkin Elmer). They use it in their long PCR kit. Tth also has RT activity,
> but I don't know that anyone has ever pushed it for length.
> 
> Sure would be nice to make an infectious cDNA clone with 1 PCR reaction. :-)
> 
> -Steve
> 




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