Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening

Chunyi George Huang, Peter C Agre, Kevin Strange, Todd Lamitina

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)83-86
Number of pages4
JournalApplied Biochemistry and Biotechnology - Part B Molecular Biotechnology
Volume32
Issue number1
DOIs
StatePublished - Jan 2006

Fingerprint

Mutagenesis
Polymerase chain reaction
Screening
Enzymes
Genes
Polymerase Chain Reaction
Gene Knockout Techniques
Essential Genes
Caenorhabditis elegans
Assays
Throughput
Personnel

Keywords

  • Ethylnitrosourea
  • Mutation
  • Polymerase chain reaction

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Isolation of C. elegans deletion mutants following ENU mutagenesis and thermostable restriction enzyme PCR screening. / Huang, Chunyi George; Agre, Peter C; Strange, Kevin; Lamitina, Todd.

In: Applied Biochemistry and Biotechnology - Part B Molecular Biotechnology, Vol. 32, No. 1, 01.2006, p. 83-86.

Research output: Contribution to journalArticle

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