Advantages of 33-P

Song Tan tan at aeolus.vmsmail.ethz.ch
Thu Mar 17 02:52:45 EST 1994


In article <1994Mar14.174839.28720 at alw.nih.gov>, Jim Owens
<jow at helix.nih.gov> wrote:
> In article <2lsmgm$mkk at usenet.INS.CWRU.Edu> Dan Diaz,
> bl275 at cleveland.Freenet.Edu writes:
> >Decomposition of 33-P to 33-S occurs at a level of a few percent a day. 
> >Since most of us use labeled dATP(alpha 33-P), this means that where 33-P
> >decomposition to 33-S occurs, the phosphodiester is converted to a
> sulfonic
> >acid ester just before dA residues:
> >       
> >          O                         O
> >  5'-dN~O-P-O~dA-3'  -----> 5'-dN~O-S-O~dA-3'
> >          O                         O
> 
> I have reasoned this way myself.  But is not the product with
> [35-S]dATP(alpha-S) that heat labile sulfate diester?  Why would this be
> more stable than one generated by 33-P decay?
> 
> Perhaps the radiodecay of phosphorus to sulfur leads to the problem and
> not heat instability of the sulfate diester?  I believe the problem also
> occurs with 32-P -> 32-S.

O.K., as Dan points out, use of [35-S]dATP(alpha-S) does not share the same
problem as [alpha-33-P]dATP because incorporation of [35-S]dATP(alpha-S)
results in phosphothiolates and not sulfate diesters. (35-S decays to 35-Cl
anyway.)

But I'm pondering the other part of Jim's posting.  As he points out, 32-P
decays to 32-S, and so use of [alpha-32-P]dATP should result in exactly the
same problem that Dan described for [alpha-33-P]dATP.  An application
besides sequencing where this might be a problem would be if one
end-labelled DNA at the 3' end by filling in a 5' overhang with Klenow and
[alpha-32-P]dATP, and then used the probe for footprinting.

Have I missed something here or is it correct that use of [alpha-32-P]dATP
will result in the same problem that Dan described for [alpha-33-P]dATP?


Song Tan
Institute for Molecular Biology and Biophysics
ETH-Honggerberg (Swiss Federal Institute of Technology)
8093 Zurich, Switzerland
email:  tan at aeolus.vmsmail.ethz.ch



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