nature of "radiolysis" of probes
dk at no.email.thankstospam.net
Fri Jan 18 23:31:07 EST 2002
Pamela Norton <pamela.norton at mail.tju.edu> wrote:
>In article <email@example.com>, Michael L. Sullivan
><mlsulliv at facstaff.wisc.edu> wrote:
>> I have a question on the nature of "radiolyis" of P-32 hybridiazation
>> probes. When people talk about probes breaking down due to radiolysis, is
>> that occuring because of
>> 1) decay of the incorporated P-32 to S-32 results in breakage of the
>> phosphodiester bond in some way
>> 2) the action of the emitted beta particals in the sample results in
>> fragmentation of the probe
>> 3) a combination of the above two processes.
>> I'm mostly curious (but also have a more practical matter in mind about a
>> failed hybridization) and haven't really found the answer anywhere else.
>I believe that #3 is correct. This belief is based on part on observing
>that 32P-labelled rNTPs and dNTP stocks become unusable much faster
>than would be accounted for by the isotope decay rate. That is, stocks
>greater than one half life old perform very poorly. On the other hand,
>33P-labelled dNTPs remain usable for months in sequencing reactions. I
>suspect that the emitted 32P beta particles are damaging the
>Any information besides this admittedly anecdotal account is welcome.
#2. P-32 decay energy is much higher (~10-fold IIRC) than that of P-33
(that's actually the only reason P-33 results in sharper bands). The very
word radiolysis implies breaking down. That's what happens when you hit
any molecule too hard too many times.
Standard, age-old recommendations to reduce radiolysis:
- Dilute the isotope as much as you can afford it.
- Add non-radioactive "carrier".
- Keep high energy compounds like P-32 at -70C
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