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Genome Res. 1999 Sep;9(9):859-67.

High-throughput isolation of Caenorhabditis elegans deletion mutants.

Author information

  • 1Axys Pharmaceuticals, NemaPharm Group, South San Francisco, California 94080, USA. lliu@cambriabio.com

Abstract

The nematode Caenorhabditis elegans is the first animal whose genome is completely sequenced, providing a rich source of gene information relevant to metazoan biology and human disease. This abundant sequence information permits a broad-based gene inactivation approach in C. elegans, in which chemically mutagenized nematode populations are screened by PCR for deletion mutations in a specific targeted gene. By handling mutagenized worm growth, genomic DNA templates, PCR screens, and mutant recovery all in 96-well microtiter plates, we have scaled up this approach to isolate deletion mutations in >100 genes to date. Four chemical mutagens, including ethyl methane sulfonate, ethlynitrosourea, diepoxyoctane, and ultraviolet-activated trimethylpsoralen, induced detectable deletions at comparable frequencies. The deletions averaged approximately 1400 bp in size when using a approximately 3 kb screening window. The vast majority of detected deletions removed portions of one or more exons, likely resulting in loss of gene function. This approach requires only the knowledge of a target gene sequence and a suitable mutagen, and thus provides a scalable systematic approach to gene inactivation for any organism that can be handled in high density arrays.

PMID:
10508845
[PubMed - indexed for MEDLINE]
PMCID:
PMC310813
Free PMC Article

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