pyrophoshatasing cell extract for nucleotide depletion?
rfwhittier at hotmail.com
Wed Aug 7 00:47:05 EST 2002
D.K. replied to Johannes Graumann:
>>If I were to make a cell extract and throw in pyrophosphatase - would I
>>manage to deplete it from nucleotide triphosphates?
>Very likely. Same is regularly done with non-specific phosphatases and
>apyrase (said to be adenine-specific but happily chews up everythting else,
Pyrophosphatase is included in some commercial DNA sequencing mixes to
prevent pyrophosphate exchange. This is when DNA polymerase runs
backwards, combining pyrophosphate with the 3' residue to remove it
from the chain. Cleaving pyrophosphate makes the extension reaction
essentially irreversable and evens out the sequencing ladder. (I assume
that this is because some ddNTPs are more susceptible to the reverse
reaction than others). However, the dNTPs are left intact; otherwise
this trick would be useless. Why should NTPs be any different?
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