Advantages of 33-P

Jim Owens jow at helix.nih.gov
Mon Mar 14 12:48:39 EST 1994


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
>
>The problem occurs when you heat the sequencing reaction at 75-80 'C in
>preparation for loading.  At this point the sulfonic esters are
hydrolyzed,
>with the result that sequence pattern is littered with bands across all
>four lanes everywhere an 'A' occurs.  Not a pretty sight, my friends,
>enough to make a biochemist cry.  The heat lability of sulfonic acid
>esters may not interfere with its use in other molecular biological
>applications, but in sequencing, 33-P has a definite disadvantage.
>
>I plan to stick to [35-S]dATP(aS).  If you will be running your reactions
>the same day, 33-P may be fine.  If you are trying to work out a
difficult

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.

Trying to work this out for myself, too.

Jim Owens



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