Structural Analyses of the Ribosome by Chemical Modification Methods - Biophysical Approaches to Translational Control of Gene Expression - page 73

4.3.3 Radioactive Primer Labeling

While radioactive primer labeling is much more versatile than end-labeling, it has
its own inherent limitations. The use of radioactivity is the first disadvantage. The
mostly commonly used radioisotope is 32 P. While 32 P provides excellent signal
strength, its relatively short half-life limits the ability to store primers for long periods
of time, thus requiring continuous relabeling of primers in order to replicate experiments.
This can become labor intensive and costly. Other radioisotopes offer longer half-
lives (such as 14 C or 35 S), although primers labeled with these isotopes have
significantly weaker signals. Additionally, autoradiolytic decay products can pro-
duce artifactual signals and increase background noise. An additional disadvantage
lies in identification of appropriate primers. For example, hybridization of primers
to A-U rich regions of RNA requires lower annealing temperatures, which can result
in nonspecific hybridization, again increasing background noise. Lastly, at the time
that this technique was first developed, primers were derived from restriction diges-
tion products, thus making it difficult to guarantee that the primer had the correct
sequence. This problem was solved with the advent of cost-effective DNA
oligonucleotide synthesis technologies.

4.3.4 Urea Polyacrylamide Gel Electrophoresis

The method of separation of the fragments is another area for improvement. For
urea polyacrylamide gel electrophoresis to be useful, large-format gels must be used
(as large as 45 cm). Unfortunately, these large gels are extremely fragile and prone
to problems with pouring and breakage. Length of run represents another potential
problem with this method. Even with extremely large gel formats, only 150-200
base pairs can be resolved with any level of accuracy. Therefore, for large RNAs
such as the ribosomal RNA or viral genomes, many gels are required to obtain full
coverage. Lastly, quantification of the autoradiographic data generated from these
gels is imprecise. Each band for which quantifiable data is desired must be assessed
by computer software with high level of human manipulation for accurate results.
The labor intensiveness of this kind of analysis led more often to simple qualitative
interpretation of results based on perceived differences in band intensities.

4.3.5 Chemical Modi fi cations

The chemicals used to modify the RNA represent another area for improvement.
While each chemical has its positives and negatives, multiple chemicals are required
to fully interrogate a region of RNA. This results in an intense time requirement to
fully interrogate large RNAs. Compounding this is the fact that many of these chemicals