Targeted Recovery of Mutations in Drosophila

Alyssa Bentley, Bridget MacLennan, Jonathan Calvo and Charles R. Dearolf

ABSTRACT

Reverse genetic techniques will be necessary to take full advantage of the genomic sequence data for Drosophila and other experimental organisms. To develop amethod for the targeted recovery of mutations,we combined an EMS chemical mutagenesis regimen with mutation detection by denaturing high performance liquid chromatography (DHPLC). We recovered mutant strains at the high rate of z4.8 mutations/ kb for every 1000 mutagenized chromosomes from a screen for new mutations in the Drosophila awd gene. Furthermore, we observed that the EMS mutational spectrum in Drosophila germ cells shows a strong preference for 59-PuG-39 sites, and for G/C within a stretch of three or more G/C base pairs. Our method should prove useful for targeted mutagenesis screens in Drosophila and other genetically tractable organisms and for more precise studies of mutagenesis and DNA repair mechanisms.

Genetics 156: 1169–1173 (November 2000)