long PCR with dUTP

Dr. Duncan Clark duncan at genesys.demon.co.uk
Tue Feb 25 04:23:02 EST 1997


In article <mcgorry-2402971920360001 at quadra950.pathology.pitt.edu>,
"Michael C. Gorry" <mcgorry at med.pitt.edu> writes
>I am trying to amplify a longer DNA (4kb) fragment using dUTPs instead
>of dNTPs.  I have been unable to get an amplification product with U but
>have obtained it with T. Is there a limit on the amount of U that can be
>incorporated?  I have not been able to find anything out about it and before
>putting more effort into it, I would like to know if anyone has any information.
>To date, I have tried taq, klentaq, pfu, and rTth.  I had expected the best
>results with the rTth, but was unable to reach the 0.6kb control with any of the
>polymerases used.
>
None of the proof-reading pols will PCR using dUTP ie Pfu, Pwo, Vent,
Deep Vent etc. This also applies to mixes using these pols. Taq uses
dUTP well but I've never tried 4kb, only up to 1kb. Do you have to have
100% dUTP, can you get away with a mix of dUTP + dTTP ie 25% dUTP. 

Duncan 

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Duncan Clark
DNAmp Ltd.
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