Eric Kofoid kofoid at biology.utah.edu
Tue Nov 29 11:50:55 EST 1994

In article <3453506wnr at genesys.demon.co.uk>, Duncan at genesys.demon.co.uk wrote:

-In article: <3abpup$a2n at dns1.NMSU.Edu>  smori at nmsu.edu (Shahram Mori) writes:
-> Steve Wylie (wylie at CSUVAX1.MURDOCH.EDU.AU) wrote:
-> : Has anyone tried RT/PCR from RNA of 10Kb or longer? I have been trying to 
-> : do this with the potyvirus (BYMV) I work with but with no success. 4Kb is 
-> : no problem providing the RT reaction is allowed to proceed for 3-4 hr. I 
-> : suspect the RT to be the limiting step so have tried M-MLV and AMV RT's 
-> : with no luck. I don't use a special long PCR kit, I've just added some 
-> : Pfu to the Taq and 8% glycerol and 1.5% DMSO to the mix, as well as 
-> : reducing to denaturation time to 10sec. Any comments gratefully accepted. 
-> : Steve Wylie, Murdoch University, Western Australia
-> I would run a gel of your RT reaction( put a film on it) to make sure that 
-> You have no RT reaction. It might ( and I suspect it is ) your PCR.
-> For denaturing such a large fragment you need at least a minute of
-> denaturation.
-I am not sure about this length of denaturation. The protocols for LA 
-PCR actually insist on short denaturation times of the order of just a 
-few seconds for 30-40kb targets, the hypothesis being that heat plus 
-the alkaline buffer in the PCR depurinates the DNA rapidly during 
-denaturation. This in turn stops the polymerase extending.
-My mind's made up. Don't confuse me with the facts!

Low pH - not high pH - depurinates DNA. On the other hand, alkaline
conditions *will* hydrolyze RNA. The longer the molecule, the greater the
probability of a break. This is probably the rate limiting step.

The following two accessible and often cited papers answer most questions
raised about long and accurate PCR:

Barnes, W.M., "PCR amplification of up to 35-kb DNA with high fidelity and
high yield from lambda bacteriophage templates", PNAS 91, 2216-2220, 1994.

Cheng, S., Fockler, C., Barnes, W.M. & Higuchi, R., "Effective
amplification of long targets from cloned inserts and human genomic DNA",
PNAS 91, 5696-5699, 1994.



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