Advantages of 33-P

Brian Smith-White smithwhi at students.msu.edu
Thu Mar 17 19:28:00 EST 1994


In Article <1994Mar14.174839.28720 at alw.nih.gov> "Jim Owens <jow at helix.nih.gov>" says:
> 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
> 
	About 30 years ago people were studying mutagenesis by radioactivity
and they were able to show that the rate of chain breakage for 32P labeled 
DNA was about twice the decay rate - one break from the decay, one break from
the beta particle (it is called ionizing radiation for a reason). I forget 
the numbers for weak beta particles. There is also the parameter of half
life. Shorter half life has more decay events per unit time.
Brian Smith-White



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