Analysis with GeneChip Microarrays
Whole genome expression monitoring will have extraordinary impact on
clinical diagnosis and therapy and bring new power to both basic
research and clinical medicine. As the field progresses we will
identify new probes for cancer, infectious disease, inherited disease,
DNA genetic damage, analysis of gene expression, and analysis of
protein expression. Equally important will be new therapeutic tools in
the form of recombinant gene products, novel drug targets, rational
drug design, and gene therapy. Next-generation efforts will allow us to
link gene expression patterns with formal characteristics of disease
models including histological, pathological, and clinical state
descriptions.
Affymetrix, Inc. has pioneered techniques for the photolithographic
synthesis of oligonucleotide arrays at very high density on silicon
chip surfaces (Pease et al., 1994) . The Affymetrix system can analyze
either expressed mRNA or DNA gene sequences (Lipshutz et al., 1999) .
Recent improvements in the production of new chips with 25-mer versus
previous 16-mer oligonucleotides and with much less chip-to-chip and
position-to-position variation than was previously observed. The
procedure is to purify RNA or DNA from the biological material to be
analyzed and subject it to reverse transcription/cRNA transcription or
direct PCR, respectively, with the incorporation of fluorescent tags in
the final steps of probe labeling. Carefully controlled hybridization
and analysis of fluorescent intensities at each position allows
abundance estimation with discrimination of specific and non-specific
signals sufficient to detect single base mismatches. The detection of
known mutations and alleles, an attractive capability of the Affymetrix
system, is applicable to several studies underway here, such as the
detection of p53 or BRCA1 mutations or p450 gene alleles that account
for slow and rapid drug metabolizers. A particularly powerful
application of sequence-specific allelic analysis is to detect
resistance alleles in infectious microbial organisms such as
tuberculosis and HIV, or the identification of microbial species using
conserved regions such as ribosomal genes. A new capability of the
Affymetrix system is a multiplex PCR and custom chip technology that is
able to "interrogate" 5200 known regions of the human genome that
exhibit single nucleotide polymorphisms (SNPs)(Hacia et al., 1999)
This approach to indivudual genotyping is likely to be capable of
providing excellent data for linkage analysis. Its genome mapping power
compared to the approach of using conventional microsatellite markers
is not yet clear. However, with so many markers able to be interrogated
on a single chip in parallel fashion it may well prove to be quite
practical and cost effective.
In a clinical context, expression profiling using the Affymetrix
system will do more than provide a better understanding of biological
and pathobiological processes and the possible function of genes and
proteins. Tumor types should be exquisitely diagnosed and should lead
to optimization of therapeutic approaches. Specific tailoring of
therapeutic approaches should be accomplished using expression
profiling in individual patients. Structure-function analysis of new
drugs may be vastly accelerated, particularly for the optimization of
therapeutic index and specificity. This may be accomplishable by
application of the genechip system to many biological systems including
microbial cultures, yeast 2-hybrid, cell culture, or in vivo models.
A typical experiment would use between 2 and 20 gene chips for a
complete study. The data provided in such an experiment can fuel
further research efforts in a laboratory for a year or more.
