Parallel analysis of genetic selections using whole genome oligonucleotide arrays

Abstract

Thousands of genes have recently been sequenced in organisms ranging from Escherichia coli to human. For the majority of these genes, however, available sequence does not define a biological role. Efficient functional characterization of these genes requires strategies for scaling genetic analyses to the whole genome level. Plasmid-based library selections are an established approach to the functional analysis of uncharacterized genes and can help elucidate biological function by identifying, for example, physical interactors for a gene and genetic enhancers and suppressors of mutant phenotypes. The application of these selections to every gene in a eukaryotic genome, however, is generally limited by the need to manipulate and sequence hundreds of DNA plasmids. We present an alternative approach in which identification of nucleic acids is accomplished by direct hybridization to high-density oligonucleotide arrays. Based on the complete sequence of Saccharomyces cerevisiae, high-density arrays containing oligonucleotides complementary to every gene in the yeast genome have been designed and synthesized. Two-hybrid protein-protein interaction screens were carried out for S. cerevisiae genes implicated in mRNA splicing and microtubule assembly. Hybridization of labeled DNA derived from positive clones is sufficient to characterize the results of a screen in a single experiment, allowing rapid determination of both established and previously unknown biological interactions. These results demonstrate the use of oligonucleotide arrays for the analysis of two-hybrid screens. This approach should be generally applicable to the analysis of a range of genetic selections.

Figure 1

Strategy for identifying sequences after a genetic selection. Rather than individual purification and dideoxy sequencing, all clones are pooled from plates and plasmid DNA is isolated in a single purification. PCR amplification using primers with 3′ sequence corresponding to vector sequence is used to selectively enrich for insert DNA from the plasmid pool. Amplified insert DNA is fragmented with DNase I, labeled with biotin-ddATP, and hybridized to an array containing oligonucleotide probes for every gene in the yeast genome.

Figure 2

Fluorescence images of a high-density oligonucleotide array containing 25-mer probes for nearly every gene on Saccharomyces cerevisiae chromosomes 5–10. (a) Fluorescence pattern obtained after hybridization of 11 control genes: YEL002c, YEL003w, YEL005c, YEL006w, YEL018w, YEL019c, YEL021w, YEL024w, YHL014c, YHL045w, and YHL044c. Dark areas correspond to probes for genes not present in the control pool. (b) A close-up view of gene YHL014c shows the exact probe features that hybridize to the insert. Red grid highlights all probe features for YHL014c. Top row of probe elements contains oligonucleotides perfectly complementary to gene sequence, whereas bottom rows contain a mismatch in the central position of the oligonucleotide. Approximate locations of complementary oligonucleotide probes along the YHL014c ORF are also shown.

Figure 3

Fluorescence image of a portion of a high-density oligonucleotide array containing 25-mer probes to nearly every gene on Saccharomyces cerevisiae chromosomes 5–10 after hybridization of YMR117c two-hybrid sample. The three lighted strips correspond to probes covering nucleotides 156–654 of ORF YER018c, nucleotides 1860–2484 of YER032w, and nucleotides 4092–4452 of YGL197w. Terminal probes are described as the most 5′ nucleotide of the most 5′ probe and the most 3′ nucleotide of the most 3′ probes that gave a positive signal. Dark areas correspond to probes for genes not present after genetic selection.