title: Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening.
abstract: The ability to generate null mutants is essential for studying gene function. Gene knockouts in Caenorhabditis elegans can be generated in a high throughput manner using chemical mutagenesis followed by polymerase chain reaction (PCR) assays to detect deletions in a gene of interest. However, current methods for identifying deletions are time and labor intensive and are unable to efficiently detect small deletions. In this study, we expanded the method pioneered by Wei et al., which used the thermostable restriction enzyme PspGI and tested the usefulness of other thermostable restriction enzymes including BstUI, Tsp45I, ApeKI, and TfiI. We designed primers to flank one or multiple thermostable restriction enzymes sites in the genes of interest. The use of multiple enzymes and the optimization of PCR primer design enabled us to isolate deletion in 66.7% of the genes screened. The size of the deletions varied from 330 bp to 1 kb. This method should make it possible for small academic laboratories to rapidly isolate deletions in their genes of interest.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Chunyi George
authors.initials: CG
authors.lastname: Huang
authors.forename: Peter
authors.initials: P
authors.lastname: Agre
authors.forename: Kevin
authors.initials: K
authors.lastname: Strange
authors.forename: Todd
authors.initials: T
authors.lastname: Lamitina
mesh_terms: D000477:Alkylating Agents / Q000633:toxicity; D000818:Animals; D017173:Caenorhabditis elegans / Q000187:drug effects / Q000235:genetics*; D018509:DNA, Helminth / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005038:Ethylnitrosourea / Q000633:toxicity*; D017353:Gene Deletion; D017238:Genes, Helminth / Q000235:genetics; D016296:Mutagenesis / Q000187:drug effects* / Q000235:genetics; D009154:Mutation / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
chemical_list: D000477:Alkylating Agents; D018509:DNA, Helminth; C083548:Tsp45I endonuclease, Thermus sp. YS45; C075307:endodeoxyribonuclease TfiI; C046545:CCWGG-specific type II deoxyribonucleases; C039566:CGCG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; D005038:Ethylnitrosourea