Worm Breeder's Gazette 15(4): 17 (October 1, 1998)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Washington University School of Medicine, St. Louis MO 63110
Only a small number of useful mapping markers and genes have been identified in the region between egl-29 and unc-52 on chromosome II. To identify polymorphisms in this region, we employed a variation of the method described in the accompanying abstract, and identified eight PCR-based polymorphisms between the wild type strains N2 and RC301. Six of these manifest as mobility shifts; the remaining two are visualized by restriction digest.
To identify polymorphisms, we designed 24mer or 25mer oligonucleotides predicted to generate approximately 1-1.5 kb fragments, based on sequence provided by the Genome Sequencing Center. Each oligo was designed from non-coding regions, and verified as non-redundant sequence. Oligonucleotide primers were tested against the N2 and RC301 templates. When there was no apparent shift between relative PCR products, or when the difference was only slight, the PCR products were digested with two or three restriction enzymes. (Seven polymorphisms were identified by this approach. The eighth was identified by sequencing the N2 and RC301 PCR products, which revealed a polymorphism that generated loss of a restriction site.) As a control, the RC301 strain was backcrossed against a rol-1 unc-52 strain (in an N2 background). Rehomozygosed wild type animals then were used as templates for PCR, to ensure that the polymorphisms segregated as predicted.
Using this approach, one third (8/24) of the oligo pairs we tested revealed polymorphisms, and almost all of these (6/8) represent deletions or an insertion that can be detected by electrophoresis. (One pair failed to generate any product, and the remaining 15 did not reflect any polymorphisms.) In sum, 6/24 primer pairs on chromosome II R revealed N2 and RC301 products with size differences detectable by gel electrophoresis, compared to 1/19 primer pairs on chromosome X LC (see accompanying abstract). Perhaps the higher incidence of deletion and insertion polymorphisms on the right arm of chromosome II correlates to the dearth of transcripts in this region; alternatively, these genomic differences could be unique to this area of the genome.
The following describes the polymorphisms we identified, including cosmid locus and size of the PCR products, and indicates the appropriate restriction enzyme to visualize the distinctions between those N2 and RC301 products that are identical or close in size. For additional information or reagents, please contact MLN or AMS.