Mark Chee

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 soon. Sequence information alone, however, is insufficient for a full understanding 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 important to have experimental tools for the direct monitoring of large numbers of mRNAs in parallel. We have developed an approach that is based on hybridization to small, high-density arrays containing tens of thousands of synthetic oligonucleotides. The arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide chemistry. RNAs present at a frequency of 1:300,000 are unambiguously detected, and detection is quantitative over more than three orders of magnitude. This approach provides a way to use directly the growing body of sequence information for highly parallel experimental investigations. Because of the combinatorial nature of the chemistry and the ability to synthesize small arrays containing hundreds of thousands of specifically chosen oligonucleotides, the method is readily scalable to the simultaneous monitoring of tens of thousands of genes.

Accessing genetic information with high-density DNA arrays

Rapid access to genetic information is central to the revolution taking place in molecular genetics. The simultaneous analysis of the entire human mitochondrial genome is described here. DNA arrays containing up to 135,000 probes complementary to the 16.6-kilobase human mitochondrial genome were generated by light-directed chemical synthesis. A two-color labeling scheme was developed that allows simultaneous comparison of a polymorphic target to a reference DNA or RNA. Complete hybridization patterns were revealed in a matter of minutes. Sequence polymorphisms were detected with single-base resolution and unprecedented efficiency. The methods described are generic and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability.

Detection of heterozygous mutations in BRCA1 using high density oligonucleotide arrays and two–colour fluorescence analysis

The ability to scan a large gene rapidly and accurately for all possible heterozygous mutations in large numbers of patient samples will be critical for the future of medicine. We have designed high–density arrays consisting of over 96,600 oligonucleotides 20–nucleotides (nt) in length to screen for a wide range of heterozygous mutations in the 3.45–kilobases (kb) exon 11 of the hereditary breast and ovarian cancer gene BRCA1. Reference and test samples were co–hybridized to these arrays and differences in hybridization patterns quantitated by two–colour analysis. Fourteen of fifteen patient samples with known mutations were accurately diagnosed, and no false positive mutations were identified in 20 control samples. Eight single nucleotide polymorphisms were also readily detected. DNA chip–based assays may provide a valuable new technology for high–throughput cost–efficient detection of genetic alterations.