EXPRESSION MONITORING BY HYBRIDIZATION TO HIGH-DENSITY OLIGONUCLEOTIDE ARRAYS

The human genome encodes approximately 100,000 different genes, and at least partial sequence information for nearly all will be available s oon. Sequence information alone, however, is insufficient for a full u nderstanding of gene function, expression, regulation, and splice-site variation. Because cellular processes are governed by the repertoire of expressed genes, and the levels and timing of expression, it is imp ortant to have experimental tools for the direct monitoring of large n umbers of mRNAs in parallel. We have developed an approach that is bas ed on hybridization to small, high-density arrays containing tens of t housands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a comb ination of photolithography and oligonucleotide chemistry. RNAs presen t at a frequency of 1:300,000 are unambiguously detected, and detectio n is quantitative over more than three orders of magnitude. This appro ach provides a way to use directly the growing body of sequence inform ation for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize sm all arrays containing hundreds of thousands of specifically chosen oli gonucleotides, the method is readily scalable to the simultaneous moni toring of tens of thousands of genes.