Whole Genome Analysis of C. elegans Using DNA Microarrays

Abstract

We have recently received funding to produce C. elegans whole genome DNA microarrays, which will be used to investigate gene expression patterns on a global scale. We are taking advantage of the C. elegans genome sequence and the software developed at the Sanger Centre to predict PCR primer pairs that will specifically amplify 1 kb coding regions from each of the predicted 18,000 C. elegans genes. The development of primers is being performed in collaboration with Stuart Kim (Stanford, CA). The DNA microarrays will be produced following protocols originally developed in the laboratory of P. Brown (Stanford, CA) and used by S. Kim to develop a partial worm DNA microarray. The microarrays will be generated by robotically gridding each of the 18,000 1kb DNA fragments onto poly-L-lysine coated glass slides. The microarrays will then be used to compare steady-state RNA populations expressed by animals with different genetic backgrounds or physiologies by differential hybridisation of cDNAs. These cDNAs will carry either a Cy3 or Cy5 fluorescent tag to be incorporated during reverse transcription. Following hybridisation, the resulting fluorescent signals will be detected by confocal laser scanning and expressed as two-colour ratios of differential expression. Bioinformatic methods for archiving and analysing these data will be extended during the course of these studies. The power of DNA microarray technology is two-fold: DNA microarrays yield functional information on genes in the form of expression profiles, and they enable an entire genome to be searched in a single reaction to identify primary and secondary response genes. Quantitative data can be obtained on both up- and down-regulated genes to help decipher genetic pathways. The technology is sufficiently sensitive to detect 2-fold differences in gene expression. We plan to use the microarrays to address a series of key questions in developmental biology. For example, we plan to compile a profile of downstream targets for each of the predicted homeodomain proteins in the worm. Finally, we will make this technology available to the members of the research community.