Targeted recovery of mutations in drosophila

Reverse genetic techniques will be necessary to take full advantage of the genomic sequence data for Drosophila and other experimental organisms. To d evelop a method for the targeted recovery of mutations, we combined an EMS chemical mutagenesis regimen with mutation detection by denaturing high per formance liquid chromatography (DHPLC). We recovered mutant strains at the high rate of similar to4.8 mutations/kb for every 1000 mutagenized chromoso mes from a screen for new mutations in the Drosophila and gene. Furthermore , we observed that the EMS mutational spectrum in Drosophila germ cells sho ws a strong preference for 5'-PuG-3' 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.