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Chemically Modified Taq - Taq Polymerase (Dec/07/2006 )

hello,
i have recently heard about this chemiclaly modified taq polymerase, that has some changes to the amino acid done so as to make it inactive. i wouldliek to knwo more details about this, if anybody knows anything or has a good reference paper to go along with it, it would be great.


thekid

-thekid-

QUOTE (thekid @ Dec 7 2006, 05:45 AM)
hello,
i have recently heard about this chemiclaly modified taq polymerase, that has some changes to the amino acid done so as to make it inactive. i wouldliek to knwo more details about this, if anybody knows anything or has a good reference paper to go along with it, it would be great.


thekid



Here, some information:
Chemically modified recombinant Taq DNA polymerase
The enzyme is a highly purified form of recombinant Taq polymerase that has been chemically modified by the addition of heat-labile blocking groups to specific amino acid residues. Prior to PCR, in its inactive state it is incapable of extending primer-dimers or mis-annealed primer-template species that form below the specific annealing temperature. The 95°C incubation step therefore serves to activate the enzyme and also to ensure a completely "clean" initial PCR cycle.


Sharkey DJ, Scalice ER, Christy KG Jr, Atwood SM, Daiss JL.
Antibodies as thermolabile switches: high temperature triggering for the polymerase chain reaction.
Biotechnology (N Y). 1994 May;12(5):506-9.

-aztecan princess-

I have been trying to make it for the last three weeks.
I have succeeded, however after one week the modified TAQ lost its efficiency (it died on me blink.gif ).
I was storing it at 4C...so next time i will be adding glycerol and keeping it in the freezer.
If anyone else on this site is succeeding please send your tips my way wink.gif

Basically what you do is the following:
Make TAQ...and dissolve TAQ pellet in 2% sucrose
Dry this soln in a vacuum-drier, down to the crystals
Resuspend in T-MBE and citraconic anhydride, which modifies the the TAQ's lysine grps
Wash with alot of hexane
Dissolve in storage buffer = modified TAQ that needs 95C for 15min to reactivate.

-carlchurchill-

see the patent description at http://www.patentstorm.us/patents/6479264-description.html

-tfitzwater-