"LONG" RT/PCR

a-library library at nimr.mrc.ac.uk
Tue Dec 13 13:58:31 EST 1994


>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

Hi to all those interested in the great RT/PCR debate! I just managed to get into
this system for the first time today, so you'll have to excuse the 'first-timers'
enthusiasm.

First, Steve,  ITS BEEN DONE! An infectious transcription clone was
generated by PCR from first strand cDNAs to CMV (cucumber mosaic virus)
RNAs by Fred Boccard, then of the John Innes Institute. ok, ok, so the RNAs are
less than 3kb, but still. The generation of the clones was described in 
Gene 114(2) 223-227 and, just to prove that they really were useful, Fred went on to 
make an enormously detailed examination of the subgenomic promoter which he
reported in Virology 193(2) 563-578. If anyone wants further info on the
technique Fred can be found at Boccard at cgeugu52.bitnet.

This may not be the appropriate forum for non-PCR approaches to
infectious clones, but has anyone considered using Sequenase to make
the second strand ? This is not as mad as it sounds at first; if good long
first strand cDNAs can be made (and they can,as others have pointed
out, be labelled and checked out on alkaline agarose gels) and purified 
(I used alkaline sucrose density gradients to get ~7kb first strand cDNAs
to pea early browning virus), and their 5' sequence is known, then it should 
be possible to anneal an oligo and use Sequenase, which is massively
more processive than any other enzyme available, to complete the second
strand. Problems with secondary structures can, so long as enough glycerol
is included in the reaction, be at least alleviated by elevated temperatures. 
Sure the enzyme wouldn't last long; but at the rate Sequenase polymerises
it wouldn't need to! Well, so goes the theory anyway. If I remember right
the only one to believe it enough to try was Simon Santa Cruz,now working 
in T Mike A Wilson's lab at the Scottish Crop Research Institute in (er) Scotland - 
Invergowrie, I think.

As to the RT of choice,when I was battling to make cDNAs to pea early browning RNAs
(~3.5 and ~7 kb) I got more and longer cDNAs with AMV RT at elevated temperatures 
than I ever did with Moloney RT (elevated temperatures meant sticking the reaction in a waterbath
at 42 C and immediately turning the bath up to 55 C....the bath took about
90' to get to that temperature,at which point the reaction was stopped and the cDNAs purified
on alkaline sucrose density gradients).

Has anyone tried the ligation-free cloning technique that Clontech is 
selling as its "PCRDirect" system? Any comments?

Matthew

 



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