John M. S. Bartlett.pdf - Bio-Nica.info

220 Bartlett
Even once novel regulatory changes are identified using differential display techniques,
it remains common to double-check these changes using a second system, such
as representational differential analyses (RDA), PCR. or Northerns, to confirm the
result. This is an important confirmatory step before investing significant effort into the
study of novel genes or gene transcripts identified by differential display (14). Even
with these caveats, differential display has already proven itself as a highly valuable
system for the study of gene expression changes during neoplastic progression.
There are many similarities between differential display technologies and serial
analysis of gene expression (SAGE). Indeed, SAGE might be seen as a logical progression
from differential display. Both use degenerate primer sequences to produce a
fingerprint of mRNA species for analysis. In differential display. these sequences may
range from the highly selective (members of a particular gene family) to the more
inclusive (polyT based) (14–21). Both methods use tagged primers, differential display
for the purpose of detection, and SAGE for the purpose of capture and ligation. The
fundamental difference between these approaches is that differential display continues
to use a semiquantitative measure of expression and although more sensitive than
microarrays in the detection of low copy number changes in expression, signal strength
remains a determining factor of sensitivity. Again, identification of transcripts with
altered expression relies on further analysis, either by sequencing of a representative
clone or by blotting with a selective probe. A strength of this approach is illustrated
by its ability to be targeted at specific genes or gene families. However, the number
of transcripts able to be analyzed is limited by the electrophoretic separation required
for the analysis of the results. Although this still has the capacity to recognize many
hundreds of distinct transcripts, it is unlikely that the capacity of differential display
can match that of either gene microarrays or SAGE. This section includes two different
differential display protocols, one using targeted primers (AU Motifs) and the other
being a more global protocol. Further techniques are available in an associated volume
in this series (13).
2.2. Microarrays
DNA Chips or microarrays are becoming much more widely applied to the investigation
of both model systems and whole tissues (14,22–25). However, these approaches
are not, strictly speaking PCR related and therefore we have included an overview of
arrays purely for completeness. DNA microarrays have the advantage of being data
rich, that is to say it is possible to analyze many thousands of genes simultaneously.
Using this approach it is possible to identify transcripts that are markedly upregulated
or downregulated after experimentation or, indeed, as recently reported in the simple
classification of cancers. This approach, in common with many conventional methods
of gene expression analysis (northerns, RDA, etc.) relies on the measurement of
signal intensity resulting from nucleic acid hybridization. Therefore, the efficiency
of detection is a compound of the efficiency of labeling and hybridization of the
individual clone. The resulting data give a semiquantitative estimate of changes in
expression either up or down.
The major weakness of the microarray approach is that it is firstly a semiquantitative
approach and that it is therefore not optimal for the detection of low copy number
gene transcripts. The major strength is the large number of genes (10 3 –10 4 ) that