Tth polymerase : Summary to date

[mwgaunt at molbiol.ox.ac.uk]

     Dear all,
              following my request in article 1993Jul12.164904.l at ox.
     ac.uk;

     >    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
     included.
    
     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 
     holding temperature.
     The RT activity coupled with its requirement for Mn++ buffer is less
     appealing.

     Any further contributions would be very welcome.

                        Many thanks to all those mentioned,
       
                                       Mike Gaunt (mwgaunt at ac.ox.uk )
 
                                       Inst. Virology
                                       Oxford UK.
                                       Tel. (0)865 512361
                                       Fax. (0)865 59962
     
            
     Company addresses:
     
     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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