High-resolution mutation detection in Caenorhabditis elegans mutants and natural isolates using array comparative genomic hybridization

An essential requirement of genetic research is the ability to identify mutations. Forward genetic screens begin by selecting for a phenotype and proceed to search for the causative mutation. Reverse genetics experiments first identify the mutation and then seek to derive the mutant phenotype, if any. Both approaches depend on efficient means of detecting mutations. This thesis describes the development of methods to facilitate the detection of mutations in the model organism, Caenorhabditis elegans, using array Comparative Genomic Hybridization (aCGH). Exon-centric oligonucleotide microarrays targeting specific chromosomes and the whole genome were designed and used to detect both large multi-gene and small single-gene deletions. Both homozygous and heterozygous deletions were identified using this technique. I showed that even single nucleotide transitions and transversions are detectable when using microarrays with sufficient probe densities, which are achievable with target regions of two Mbp or less. I also used aCGH to detect extensive natural gene content variation between the N2 Bristol strain and twelve wild C. elegans isolates. Most of the DNA copy number alterations in these strains are deletions relative to Bristol. Over 5% of the genes present in the Bristol strain are absent in at least one of the natural isolates that were examined. This represents a significant increase in the number of genes with known null alleles. These deletions were then used to infer relationships among the natural isolates, which proved to be complex. The methods described in this thesis will greatly assist in the identification of mutations in C. elegans and are also applicable to other organisms with sequenced reference genomes.Medicine, Faculty ofMedical Genetics, Department ofGraduat