Thermostable Pyrophosphatase

Dr. Duncan Clark news at hgmp.mrc.ac.uk
Mon Jun 25 03:52:21 EST 2001


In article <3B372290.11729.37AE9F at localhost>, the eminent Wolfgang
Schechinger at Academia Sinica wrote
>I discovered in NEB's catalogue a thermostable 
>pyrophosphatase. What is it useful for?

Cycle sequencing.

A standard polymerase reaction is:


                 pol + Mg
template + dNTPs --------> template-dNMP + PPi


In cycle sequencing you start with a large amount of template and
although it is only linear amplification you generate a lot of extended
template and even more PPi. The PPi then drives the reaction backwards,
inorganic pyrophosphorolysis, leading to degradation of the newly
extended template, even removing the ddNTP. You get missing bands in
your sequence.

Standard ABI or APB cycle sequencing kits are formulated with
thermostable PPase, from Thermus thermophilus or Thermoplasma
acidiphilum.

Another usage is Promega's new mutation detection system, which for the
life of me I can't remember the name of. It is based totally around
pyrophosphorolysis which occurs only if there is a probe match with a
SNP. The PPi produced is converted to ATP and luciferase does the rest.
Very neat.

Another use, although not a thermophilic enzyme, is in high yield
transcription kits. T7, T3 and SP6 RNA pols are inhibited by PPI so by
removing that with yeast PPase with appropriate Mg and NTPs, you get a
marked increase in transcribed product.

Roche diagnostics showed that by adding Tth PPase to Pwo PCRs they could
improve the product yield for one reaction. (Shown in the Tth PPase
product sheet .pdf file). 

Duncan


-- 
It might look like I'm doing nothing, but at the cellular level I'm really
quite busy.

Dr. Duncan Clark
GeneSys Ltd.
Tel. +44 (0) 1252376288
FAX +44 (0) 8701640382




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