S35 or P33 for DD-RTPCR
Bryan L. Ford
fordb at bcc.orst.edu
Sat Dec 13 21:46:15 EST 1997
> We are looking to employ DD-RTPCR to identify differential genes from a
> fungal cell. We have extracted highly pure total and mRNA, typically
> OD260 = 1.8. The integrity also looks good when run on a 1.0% TBE
> non-denaturing gel. i.e full smear and distinct ribosomal bands at the
> correct relative intensities.
> We are now ready to embark on the treacherous trail of DD-RTPCR. Having
> no previous experience with such a technique we would like to know what
> isotope to utilize. S35 dCTP or P33dATP?
> S35 dCTP apppears to be the most attractive because it less active than
> P33 which means it is easier to work with and it is CHEAP. Almost 1/3 the
> cost of p33. However, I want to be sure I can see rare products on the
> gel. I have also heard that S35 gets everywhere because of aerosols
> generated during the PCR reaction
> P33dATP has the advantage that it is more active than S35 so the signal is
> more intense and it can be monitered using a Geiger counter. The downside
> is that it costs three times more than S35.
> Therefore, can anyone suggest which isotope I should use bearing in mind
> there will be mistakes and we have not optimised the PCR conditions. We
> are using the RNAimage kit by GenHunter Corporation.
Perhaps this will help a bit. 35S will very likely cause some volatiles
in your PCR reactions, this is in the literature within the last couple
of years. For example, in Biotechniques there was a very nice assay of
the escaped volatiles from 35S sequencing, using if I recall correctly,
lead acetate impregnated paper laid over the tops of the "sealed" PCR
tubes. The lead acetate can capture both hydrogen sulfide and sulfur
dioxide with high affinity. The amount of 35S volatiles that were
captured during typical thermocycling was substantial and impressive
enough to cause me to quickly shift to 33P, in spite of the costs.
Besides the lower cost, there may be one other advantage of 35S: if your
are experimenting around and don't have your methodology well
established it may be useful to use 35S in spite of the volatiles since
the 87 day half-life of 35S is about 3X that of 33P, giving you a much
longer time to fiddle around getting things up and running. If you
decide to go with 35S, watch out if you place your PCR machine in a
hood, the air flow across the block(s) can set up unwanted temperature
gradients that will skew your annealing plans. The lower energy beta
(about one half of 33P) gives you only a bit more safety even if you are
working with larger amounts of curies, and I would think no significant
safety factor advantage when weighed against the very substantial
volatility risks of 35S.
Another facter to consider is the much poorer ability of some enzymes to
utilize 35S-ontaining nucleotides. I have personally seen a dramatic
inability of several thermostable polymerases to extend a primer with
such nucleotides. I suspect that kinasing might be problematic as well.
These questions are happily moot with 33P.
Hope this helps. Good luck,
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