What happens to 32P-labelled compounds?

Tom Schneider toms at fcs260c2.ncifcrf.gov
Fri Jul 31 18:05:26 EST 1992


In article <31JUL199206463931 at aardvark.ucs.uoknor.edu>
broe at aardvark.ucs.uoknor.edu (Bruce Roe) writes:

>In article <3660 at fcs280s.ncifcrf.gov>, toms at fcs260c2.ncifcrf.gov (Tom Schneider) writes...
>>In article <1992Jul30.085812.18345 at polaris.utu.fi>
>>eepee at polaris.utu.fi (Esa-Pekka P{lvim{ki) writes:
>
>	<stuff deleted>
>
>>An interesting question on these lines is:
>> 
>>Suppose one labeled some DNA with 32P.  Then look under an STM.  Watch again.
>>Watch again.   POOF!  Look again and there is a little crater there or what?
>>This experiment is easy to do and one would be (I think) the first person
>>to observe the radioactive decay of a single molecule.  If someone does
>>the experiment I'd like to know the result.  (I ran some calculations on this
>>a while back, and it seems like one would not need to wait very long to see
>>some molecules decay if one watched a large enough set of them.  Half life
>>of 32P is only 2 weeks.)
>

>IMHO you would see a 32S where there once was a 32P but *no crater*.

How do you know?  Have you done the experiment?  This is a mini-atomic
explosion.  It releases a lot of energy.  Perhaps one COULD detect damage
in the local surface!

>Lest we forget, the radiolabeled molecules we use as "tracers" usually
>contain a very large excess of non-radiolabeled carrier

Well obviously you wouldn't add any carrier!!!!  :-)  And get the hottest
stuff available.

I discussed this idea in December 1990 on sci.nanotech (clearly nobody
has done the experiment yet, maybe one of you PRACTICAL folks would be
interested to do it):

From
| the latest edition of "Molecular Cloning": a laboratory manual, second edition,
| Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory Press, 1989,
| page 10.6-10.8.  Mix together:
|   dATP   
|   dTTP
|   dGTP
|   dCTP
|     (each with 32P on the alpha position, so it stays in the final product)
|   DNA
|   buffer (with salts and water)
|   DNA polymerase
| The DNA polymerase will bind to the DNA at nicks, and run along, replicating
| the DNA, replacing the 'cold' bases with the 'hot' bases.  They say that
| one can use all four radiolabeled to get things really hot:  "By replacing the
| preexisting nucleotides with highly radioactive nucleotides, it is possible to
| prepare 32P-labeled DNA with a specific activity well in excess of 10^8 cpm/ug."
| 
| Now, that should tell us how frequently the bases are hot, if I knew what to
| calculate next.  Lesee....
|  
| The rule is if we divide the ug by the length in kb and multiply by 1.5
| we get the number of picomoles.  So we have for 1 ug 1500 pmole of bases.
| but there are about 10^23 molecules per mole, and a pmole is 10^-12 mole,
| so that's 1.5x10^11 basepairs.  Each could bust up, so that's 3x10^11 base
| pairs.  With 10^8 counts per minute, that's 3x10-4 cpm per base.  If we
| were to watch 100 bases for an hour, we should see about 2 events.
| This is a rough calculation, and I haven't checked it closely (could someone
| confirm please?) but it looks like the experiment is practical!
 
So you nick translat VERY HOT, look under STM, wait a day and look again.  Hot
DNA should break apart (or get knocked around); cold DNA should just sit
there.  One fellow made a calculation that said the "explosion" would
be quite energetic.

>A simplier experiment than Toms, would be to lable a batch of DNA with
>alpha-32P-dATP and then run a series of gels (say 1 every other day)
>and look to see if the DNA falls appart in time as the 32P goes to 32S.
>My guess is if the tube were not contaminated with nucleases you'd not
>see any degredation.

That's an interesting control experiment.  It does not replace the direct
observation of an atomic decay.

  Tom Schneider
  National Cancer Institute
  Laboratory of Mathematical Biology
  Frederick, Maryland  21702-1201
  toms at ncifcrf.gov



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