"LONG" RT/PCR

Oberste oberste at fcs260c.ncifcrf.gov
Wed Nov 23 13:31:45 EST 1994


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


-- 
M. Steven Oberste, Ph.D.                  Internet: oberste at ncifcrf.gov
Virology Division, USAMRIID
Ft. Detrick
Frederick, MD 21702-5011



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