The investigator plans to generate and identify deletion mutations in 200 genes of C. elegans in year -01, 800 more genes in year -02 and 1000 more genes in year -03 (2000 genes total) using methanesulfonate (EMS), diepoxybutane (DEB) or trimethylpsoralen/UV (TMP/UV). He will identify the locations of three deletions using an adaptation of the multiple rounds of PCR amplification that he has already used to detect deletions in 9 different C. elegans genes. The strategy is based on the ability to detect deletions at a specific target locus at a frequency of 1 in 400,000 mutagenized genomes, as depicted in three figures on pages 20-22 of the application. The approach will be to mutagenize L4 larvae, allow them to develop to F1 organisms and then distribute 12-13 F1 organisms to each well of 384 standard 96-well microtiter plates for a total of approximately 37,000 groups of 12-13 F1 organisms, representing approximately 921,000 mutagenized genomes. After growth to F2, about 40 percent of the worms will be transferred to a fresh set of 384 microtiter plates, incubated with proteinase K to release their DNA and the different DNA samples pooled from the 384 plates to a single plate (96 pooled samples total). Each of these pooled samples will be subjected to 200-300 different, nested PCR amplifications, searching for PCR products that are smaller than expected. A smaller PCR product will suggest that a deletion has occurred in the gene flanked by the specific PCR primer set. In the nine examples given, the deletions ranged between 0.8 kb and 3.0 kb. Once a potential deletion is detected, it will be verified and traced back via repeated PCR amplifications to a specific well containing one of the original 37,000 groups of 12-13 F1 organisms and eventually to the progeny of the single organism that experienced the deletion in that gene, which will then be cryopreserved. It is estimated that a total of 700 separate PCR amplifications will be necessary for each deletion that is detected and traced to the original organism. In addition, since only enough DNA will be isolated for an original 200-300 PCR amplifications, a new mutagenized library will need to be made every 2-3 months or so and replated with the 384 96-well plates. It is estimated that this plating alone will consume 3 days. When the entire process is up and running, it is estimated that a minimum of 500 PCR reactions will be conducted each day by each lab worker (2500/week).
| Moulder, Gary L; Cremona, Gina H; Duerr, Janet et al. (2010) ?-actinin is required for the proper assembly of Z-disk/focal-adhesion-like structures and for efficient locomotion in Caenorhabditis elegans. J Mol Biol 403:516-28 |
| Edgley, Mark; D'Souza, Anil; Moulder, Gary et al. (2002) Improved detection of small deletions in complex pools of DNA. Nucleic Acids Res 30:e52 |
| Friedman, L; Higgin, J J; Moulder, G et al. (2000) Prolyl 4-hydroxylase is required for viability and morphogenesis in Caenorhabditis elegans. Proc Natl Acad Sci U S A 97:4736-41 |
| Kelleher, J F; Mandell, M A; Moulder, G et al. (2000) Myosin VI is required for asymmetric segregation of cellular components during C. elegans spermatogenesis. Curr Biol 10:1489-96 |
