Michael P. Mittmann

Genome-wide expression monitoring in Saccharomyces cerevisiae

The genomic sequence of the budding yeast Saccharomyces cerevisiae has been used to design and synthesize high-density oligonucleotide arrays for monitoring the expression levels of nearly all yeast genes. This direct and highly parallel approach involves the hybridization of total mRNA populations to a set of four arrays that contain a total of more than 260,000 specifically chosen oligonucleotides synthesized in situ using light-directed combinatorial chemistry. The measurements are quantitative, sensitive, specific, and reproducible. Expression levels ranging from less than 0.1 copies to several hundred copies per cell have been measured for cells grown in rich and minimal media. Nearly 90% of all yeast mRNAs are observed to be present under both conditions, with approximately 50% present at levels between 0.1 and 1 copy per cell. Many of the genes observed to be differentially expressed under these conditions are expected, but large differences are also observed for many previously uncharacterized genes.

High-throughput polymorphism screening and genotyping with high-density oligonucleotide arrays

A highly reliable and efficient technology has been developed for high-throughput DNA polymorphism screening and large-scale genotyping. Photolithographic synthesis has been used to generate miniaturized, high-density oligonucleotide arrays. Dedicated instrumentation and software have been developed for array hybridization, fluorescent detection, and data acquisition and analysis. Specific oligonucleotide probe arrays have been designed to rapidly screen human STSs, known genes and full-length cDNAs. This has led to the identification of several thousand biallelic single-nucleotide polymorphisms (SNPs). Meanwhile, a rapid and robust method has been developed for genotyping these SNPs using oligonucleotide arrays. Each allele of an SNP marker is represented on the array by a set of perfect match and mismatch probes. Prototype genotyping chips have been produced to detect 400, 600 and 3000 of these SNPs. Based on the preliminary results, using oligonucleotide arrays to genotype several thousand polymorphic loci simultaneously appears feasible.

Deciphering Molecular Circuitry Using High-Density DNA Arrays

The immense amount of sequence data available from expressed sequence tag (EST) databases (1,2), together with the development of technologies for the highly parallel analysis of gene expression (3–5) have created the opportunity to interrogate biochemical pathways and gene function on an unprecedented scale. We describe here a set of high-density DNA arrays containing oligonucleotides complementary to more than 6,500 human EST’s. These arrays were used to generate normal and breast cancer specific gene expression profiles. More than 1,500 expressed genes were detected in both cell types examined with 85% of all gene expression observed in the range of 1–50 copies per cell. Over 300 genes demonstrated significantly different levels of expression between normal and transformed breast cells. Increased mRNA levels were observed for the Her2/neu oncogene and genes involved in its signal transduction pathway, including Grb-7, Ras, Raf, Mek and ERK. In addition, a simple categorization of the expression changes revealed patterns characteristic of loss of wild-type p53 function. Genotyping of the p53 locus using a DNA re-sequencing array revealed inactivating mutations in the p53 DNA binding domain and loss of heterozygosity. These data demonstrate a general array-hybridization-based approach to deciphering biochemical pathways and generating testable hypotheses concerning the mechanisms of cell growth and differentiation.