Methods and reagents: Use of 33-P, a safer way to sequence?
 
Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet. Since questions about laboratory safety procedures are common on the
net, this month's column discusses the use of the radioisotope 33-P as an
alternative to 32-P.

Some hazardous wastes can accumulate for years without being collected and
disposed of. Do you have stacks of radioactive waste that you'd like to pawn
off permanently onto someone else? Just how safe is your lab with such
chemicals lying about? What can you do to get rid of some of these hazards?
Well, for starters, how about switching from 32-P to 33-P?

Since the introduction of 33-P, researchers have thought that it might be worth
a try to reduce the level of radioactivity within the lab and improve the
readability of their sequencing gels all in one fell swoop. 33-P is
particularly suitable for those who would rather not be exposed to the
higher-level isotopes for long periods of time. It is prepared by irradiation
of 33-S and has physical properties between those of 32-P and 35-S. Since it is
a beta-emitter with lower energy emission than 32-P [with a maximum energy of
emission of 0.249 (MeV) as compared with 1.71 MeV], it produces a sharper image
on X-ray film than 32-P, as well as providing a less hazardous working
environment.

Because of the higher-level emission compared with 35-S (which has a maximum
energy of emission of 0.167 MeV), it is more sensitive for detecting small
amounts of labeled probe and requires less exposure time on X-ray film.
In addition, 33-P has an extended half-life of 25.4 days - 11 more days than
32-P - allowing samples to remain active for longer. [1]

There are distinct advantages to using 33-P as opposed to 32-P or 35-S for
certain applications, such as linear amplification DNA sequencing, also known
as linear polymerase chain reaction (LPCR) sequencing or cycle sequencing.
This technique is based on the extension of DNA from an annealed primer by a
thermostable polymerase in the presence of all four deoxynucleotides plus a
proportion of one dideoxynucleotide terminator. Incorporation of the
dideoxynucleotide into the growing single strand prevents further extension.
Cycling of the temperature using a PCR machine causes the terminated
single-stranded DNA to be released from the template at high temperature, so
that the next round of primer annealing and extension can occur at low
temperature.  When all four reactions, each with a different dideoxynucleotide
terminator added, are run next to each other on a polyacrylamide gel, a ladder
of bands is seen by autoradiography.

Using 33-P-labeled deoxynucleotides in the extension reaction tubes achieves
more sensitivity than 35-S, with the added advantage that the X-ray images of
larger single-stranded DNA strands at the top of the gel do not smear together,
as can be the case when using the higher-energy 32-P. Thus, more of the gel is
legible and more sequence data can be gathered. [2]

Now for the bad news: some netters who have used nucleotides radiolabeled with
33-P are beginning to rethink its use compared with other more traditional
isotopes.  The newcomer on the block (pun intended) is having some difficulty
establishing itself as the isotope of choice.  To some, it is considered a
marketing gimmick and there is some amount of dissatifaction with its
performance as compared with the other labels available, especially when used
in cycle sequencing, for which it was originally intended.

Dan Diaz (BL275@cleveland.Freenet.Edu) wrote that single-stranded sequencing
samples containing [alpha-33-P] dATP look as good as or better than those
containing 35-S when samples are run directly on a sequencing gel on the same
day. However, when the same samples are stored frozen at -20 degrees C for more
than two or three days, bands in all four lanes (BAFLs), which correspond to
the four dideoxy bases added individually, are seen wherever an adenosine
residue exists in the DNA sequence.

The cause of the aberrant banding pattern might be the destruction of
single-stranded DNA by ionizing radiation, or might be the result of stalling
of the polymerase during the extension reaction. A more plausible explanation
is that, when 33-P decays to 33-S, the phosphodiester is converted to a more
labile sulfate ester.  Heating of the sample to 70-90 degrees C before running
the gel then causes hydrolysis of the bond wherever an adenosine residue occurs
in the sequence.  Presumably, use of 33-P-labelled dCTP would result in BAFLs
where cytosine residues occur. BAFLs are usually observed nearest to the
primer, since this is as far as incorporation of label occurs when limited by
the concentration of radionucleotides. Loss of sequence data is therefore
observed higher up on the gel. This strange banding pattern should be minimal
when using end-labeled primers instead of incorporating alpha-33-P
deoxynucleotides, but using this method adds extra steps and therefore time to
the overall process.

Researchers who complete the procedure in a single day, including cycling by
PCR and polyacrylamide gel electrophoresis, have the advantage of gaining more
sequence data. However, running a gel and reading the sequence patterns at a
later date may be difficult because the DNA is destroyed over a few days.

Netters recognize this property of 33-P as a major disadvantage since, as well
as prohibiting them from doing the PCR sequencing reactions and the gel run on
different days, it also prohibits the running of subsequent gels for
confirmation of sequences or analysis of a particularly difficult stretch of
nucleotides from the same set of dideoxy-terminated reaction tubes. In order to
avoid these problems, some people avoid 33-P and instead perform quadruple
reactions (four times the amount of everything in the original reaction) using
35-S label. In this way, the reaction tubes can be stored for longer at -80
degrees C before running several gels.

A further consideration in using 33-P is that it can cost up to four times that
of the equivalent 32-P-labeled nucleotides, owing to the specialized procedure
used to create the isotope. However, other applications of 33-P may justify the
extra expense since, when 33-P is used in riboprobes for in situ hybridization
experiments, there appear to be fewer background problems than with
35-S-labeled probes. [3]

Alternatives to the hassles of radioactive waste management include using any
of the nonradioactive methods, such as chemiluminescent detection systems,
which are becoming more popular. [4] In the past, this and other fluorescent
techniques presented high background problems caused by (1) substrate, (2)
blotting material, (3) blocking agents and (4) the method of applying the
solutions to the membrane.  Further investigations are now solving some of
these problems. [5,6]

After having tried 33-P, some are now switching back to the traditional labels
of 35-S for sequencing and 32-P for hybridizations and similar experiments to
relieve the burden of doing more reactions at a higher cost.  Most feel that
35-S is safe enough and cost effective, while 32-P is cheaper and faster than
33-P. It is generally felt that, in the hands of a trained and competent
researcher, 32-P, as any radioactive isotope, can be used safely.

References:

[1] Evans, M. R., and Read, C. A. (1992) Nature 358, 520-521

[2] Adams, S. M., and Blakesley, R. (1991) B.R.L. Focus 13, 56-58

[3] Faulkner-Jones, B. E. (1993) Amersham Life Science News 11, 5-6

[4] Beck, S., et al. (1989) Nucleic Acids Res. 17, 5115-5123

[5] Martin, C., et al. (1991) BioTechniques 11, 110-113

[6] Steck, T. R. (1994) BioTechniques 16, 406-407

*******************************************************************************
Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the author.

You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/may94.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1993. Methods and reagents - Use of 33-P, a safer way to sequence?
Trends in Biochemical Sciences 19(5):227-229.

*******************************************************************************
* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
*******************************************************************************