Tth polymerase : Summary to date
mwgaunt at molbiol.ox.ac.uk
mwgaunt at molbiol.ox.ac.uk
Fri Jul 23 17:38:05 EST 1993
following my request in article 1993Jul12.164904.l at ox.
> I am seeking info on the comparative advantages or other-
> of Tth polymerase over Taq (or even Tth v. RTase ). Panaccio
> and Lew (1991) briefly report Tth being able to function in
> a greater level of blood impurities than Taq.
the summary to date is as follows.
Jon Sayers, an experienced user of PCR, informs that a student he
supervised never got Tth to work as well as Taq.
Laura Via is neither satisfied with the DNA polymerase activity of
Tth. Regarding the RT activity of Tth pol Laura writes that using the
Cetus RT-PCR kit on bovine parovirus infected cell lines (presumably
on viral mRNA, parovirus being ssDNA ) found no evidence of reverse
transcription even with kit and lab RNA controls. Standard RTases
though did work.
Andre Hamel et al have used Tth pol for the direct RT-PCR of tissue
culture lysates in deionised formamide (a very potent RNA inhibitor ).
Andre's method, cell pellet + drop PBS + >10 volumes formamide, uses
the suprising tolerance of Tth polymerase to formamide- Tth withstanding
a 1:5 formamide dilution; Taq hardly withstands a 1:20 dilution at
most. Furthermore Pfu, Vent and Deep Vent (NEB Pfu ) as well as the
exo minus versions, tolerate similar levels of formamide.
She advises that direct DNA-PCR from blood is feasible, whilst for RNA
a thorough extraction procedure is required. A highly comprehensive
protocol of their modified RNA extraction from a method described in
Nature (Macfarlane and Dahle, Mar.11, 1993, pp186 ), which uses
Catrimox 14 detergent [Iowa Biotech Corp, direct only ], acid GITC
and acid phenol, as well as useful comments and precautions have been
Juliette Faraco's reply, from work two years previous, says that initial
DNA-PCR from blood was unsuccessful using Taq, but got the results
using Tth pol. Subsequently Taq worked when the DNA was extracted with
Gene Clean, although heavy shearing will occur.
Finally Roland Rhubner highlights a DNA polymerase mix - Dynazyme
[ Finnzyme Oy] comprising of Vent, Taq and Dynazyme of ratio 1:2:10,
giving very good yields. Roland also mentions that exactly matching
primers may be required for enzymic activity, if so only known
sequences may be confidently amplified.
Thus as a routine enzyme Tth does not appear to be the best option.
With regards to the DNA polymerase activity on blood based samples
Tth is certainly worth trying, particularly with a formamide based RNA
extraction. In this respect exo minus enymes will also be compared,
exo plus enzymes being less convieniant with no PCR refridgeration
The RT activity coupled with its requirement for Mn++ buffer is less
Any further contributions would be very welcome.
Many thanks to all those mentioned,
Mike Gaunt (mwgaunt at ac.ox.uk )
Tel. (0)865 512361
Fax. (0)865 59962
Iowa Biotech Corp. Fax. (0101) 319-335-4127
Finnzymes Oy -only UK distributor known
Flowgen Instru. Ltd,
Sittingbourne Fax. (0)795 471185
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