Macrorestriction Analysis of Caenorhabditis elegans Genomic DNA

• Published in: Genetics (Austin) 1996-10, Vol.144 (2), p.609-619
• Main Authors: Browning, H, Berkowitz, L, Madej, C, Paulsen, J. E, Zolan, M. E, Strome, S
• Format: Article
• Language: English
• Subjects: Animals; Caenorhabditis elegans - genetics; Chromosomes; Deoxyribonucleic acid; DNA; Genes; Genetics; Investigations; Multigene Family; Mutation; Polymorphism, Restriction Fragment Length; Worms
• ISSN: 0016-6731; 1943-2631; 1943-2631
• DOI: 10.1093/genetics/144.2.609

The usefulness of genomic physical maps is greatly enhanced by linkage of the physical map with the genetic map. We describe a "macrorestriction mapping" procedure for Caenorhabditis elegans that we have applied to this endeavor. High molecular weight, genomic DNA is digested with infrequently cutting restriction enzymes and size-fractionated by pulsed field gel electrophoresis. Southern blots of the gels are probed with clones from the C. elegans physical map. This procedure allows the construction of restriction maps covering several hundred kilobases and the detection of polymorphic restriction fragments using probes that map several hundred kilobases away. We describe several applications of this technique. (1) We determined that the amount of DNA in a previously uncloned region is < 220 kb. (2) We mapped the mes-1 gene to a cosmid, by detecting polymorphic restriction fragments associated with a deletion allele of the gene. The 25-kb deletion was initially detected using as a probe sequences located approximately 400 kb away from the gene. (3) We mapped the molecular endpoint of the deficiency hDf6, and determined that three spontaneously derived duplications in the unc-38-dpy-5 region have very complex molecular structures, containing internal rearrangements and deletions.