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PCR conditions for DHPLC ?

Colin Dolphin colin.dolphin at kcl.ac.uk
Mon Jan 31 08:26:24 EST 2000


Dear All

We are attempting to optimise the PCR conditions for DHPLC and are trying
to find a reproducible and relatively cheap enzyme, or enzyme
combination, to minimise mismatch incorporation into resulting amplicons.
The use of PEs Amplitaq Gold is recommended and it does indeed give good
quality DHPLC peaks with minimal leading shoulders indicating low error
rates. However, as I understand it, this enzyme, albeit very useful in
hot-start PCR, does not possess any 3' to 5' proof-reading activity in
the way, say, Pfu does so why should it have any better qualities in
terms of error rates than any other (cheaper !) enzyme? Addition of Pfu
to the Amplitaq Gold has been suggested to improve fidelity but this
seems to defeat the object of using a hot-start enzyme. It appears to me
that the "best enzyme" might be a highly processive enzyme (most standard
Taqs) in combination with a dash of Pfu, or equivalent, to add
proof-reading activity together with a hot-start facility achieved,
perhaps, by adding appropriate antibodies. Does such an "enzyme" exist?
Do people doing DHPLC have any suggestions? Thanks in advance,

Colin


Colin Dolphin
Dept of Pharmacy
Kings College London
London SE1 8WA
colin.dolphin at kcl.ac.uk




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