Inactivation of Thermo Sequenase / Taq polymerase - (Aug/30/2005 )

Dear all,

We are using the Thermo Sequenase of Amersham (an engineered derivative of the Taq polymerase) for oligo end-labelling, followed by hybridisation to an oligonucleotide array. It appears that there is still some enzyme activity during the hybridisation, leading to false positive signals. Now we would like to find a way to inactivate the enzyme after the labelling reaction in order to test this working hypothesis. Purification is not a viable solution as we have a rather low amount of short oligos in a 10ul volume.

I would be grateful for any suggestion on how to inactivate Thermo Sequenase. Please note, any such information on the Taq polymerase is likely to be relevant for the Thermo Sequenase, too. I was already thinking of adding Proteinase K; trying various heavy metals. However, if someone has any experience or a definite method, please share it with me - I would be grateful for that. By the way, EDTA doesn't work, we tried that already.

Looking forward to any help,

Many thanks in advance,

Lev

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Levente Bodrossy Ph.D.
ARC Seibersdorf research GmbH
Division of Biogenetics & Natural Resources
Department of Bioresources/Microbiology
A-2444 Seibersdorf, Austria
phone: +43 50 550 3548
fax: +43 50 550 3666
email: levente.bodrossy@arcs.ac.at
web: myprofile.cos.com/bodrossy
web: www.arcs.ac.at/u/ub
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The "hot start" PCR reactions rely on an anti-Taq antibody. I don't know if it is commercially available, but it might be a solution to your problem.