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 nonspecific 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 under way 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” 5,200 known regions of the human genome that exhibit single nucleotide polymorphisms (SNPs) (Hacia et al., 1999).
This approach to individual 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 two-hybrid, cell culture or in vivo models.
A typical experiment would use between two 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.