Worm Breeder's Gazette 13(5): 18 (February 1, 1995)
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.
Zoologisches Institut der Universitat, D-50923 Koln
We are interested in phylogenetic relationships among lower invertebrates. PCR-based technology of DNA fingerprinting(1) appears to be promising, relatively cheap and time/energy saving (compared to sequencing or multilocus fingerprinting). As a pilot experiment four samples of genomic DNA from N2, a C. elegans isolate from Australia, Rhabditis tripatitas and Dugesia gonocephala (Turbellaria) were diluted up to 0,1 ng/microL. DNA was subjected to PCR using several short synthetic oligonucleotides and two different taq polymerases. The amplification protocol differs from the standard PCR conditions (2) in that only a single oligonucleotide primer is employed per PCR reaction and no prior knowledge of the genome is required. Short primers, like short oligonucleotides in multilocus fingerprinting(2), detect several DNA sites. Priming sites which are located close to one another in inverted orientation are the targets for RAPD (Random Amplified Polymorphic DNA) analysis. The technique essentially scans a genome for these small inverted repeats and amplifies intervening DNA segments of variable length. For any given template-primer-taq polymerase combination (TPPC), the amplification products are reproducible-'. Amplification products were resolved for 4 hours on I . 8% agarose gels containing EtBr. Our RAPD pattems indicate that laboratory strains due to inbreeding (2) constitute essentially homogeneous worm populations without visible differences (with this techniques) between individual worms. The closer strains are related to each other, the better their RAPD pattems should resemble each other. With the two primers tested the RAPD patterns of the two C. elegans strains appear to differ in only one band, while those of the two other species differ to a much higher extent. This finding is similar to that with our multilocus fingerprints. Depending on the chosen TPPC (mentioned above) we find different banding patterns. These probably reflect variations in the informational content(2). The observation that two randomly chosen primers (out of hundreds) allow amplification of our worm DNA is consistent with the supposition that about 80% of all RAPD primers are suitable for this type of analysis in worms(4). Our first results suggest that we can distinguish strains by their defined characteristics visualized by the fingerprint technique. The results of our dilution series indicate that DNA of one C. elegans individual is sufficient for several RAPD experiments. In conjugation with analysis of developmental features (5) we plan to use this assay to analyze phylogenetic relation ships among nematodes. In addition, we would like to explore to which extent the RAPD technology 1 is suitable for comparative studies on higher taxonomic levels.