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- Modification of DNA using aflatoxin
The first step was to make the adduct (if in fact the carcinogenic process stemmed from an adduct; for additional
hypotheses, see work by W.
Thilly in Nature Genetics), and a method to do this in vitro had previously been worked out in the laboratory
by W. Busby. So, most of the experiments in this section are incubations of aflatoxin with DNA and the development of an
enzymatic system that formed AFB1*.
- Liberation of adduct from modified DNA
Once the putative adduct had been formed, the next task was to remove it from DNA using hydrolysis
methods. A good introduction to this topic is the video Hydrolysis
Methods.
(Be sure to watch The
Early Meeting with George
Büchi first.)
- Isolation of liberated adduct using the extraction
approach
The next theoretical step is to isolate the adduct from the
hydrolysate (the hydrolyzed modified DNA). This short set of experiments
attempted using a simple extraction to isolate the
adduct.
- Isolation of liberated adduct using the precolumn
approach
This alternative to extraction attempted to isolate the adduct
using a precolumn. (Good chromatography resources can be found at http://www.chromatography-online.org/index.html.
A working knowledge of the principles underlying chromatographic systems
is essential to understand this and the following path.)
- Isolation of large (mg) quantities of adduct using the
preparative column approach
Isolating enough adduct to do structural determination was an
enormous obstacle. At the time, quantities on the order of milligrams were
needed to characterize a structure. A mistake (and the ability to turn it
to one's advantage) led to the isolation of a large quantity of pure
adduct.
- Preliminary structure determination
Several spectroscopic techniques were used to determine the preliminary structure of the
adduct. As with the previous path, a good
understanding of spectroscopy is necessary to appreciate fully the work
done in deducing the structure. An excellent spectroscopy tutorial can be
found at http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.htm
- Determination of guanine attachment site to adduct
Once the preliminary structure had been formulated, one question
remained: on which atom was the guanine attached to the aflatoxin? A
beautiful series of experiments yielded a solid answer.
- Miscellaneous or unknown
- Publication or presentation
The data were in; all that was left was to publish. Was the group
quick enough to be the first group with the adduct structure?
