Hydrobiologia 378: 33–42, 1998. R. M. O’Riordan, G. M. Burnell, M. S. Davies & N. F. Ramsay (eds), Aspects of Littorinid Biology. © 1998 Kluwer Academic Publishers. Printed in Belgium. 33 A comparison of different protocols for RAPD analysis of Littorina N. Mikhailova 1,2 & K. Johannesson 2,∗ 1 Institute of Cytology RAS, St Petersburg 194064, Russia 2 Tjärnö Marine Biological Laboratory, S-452 96 Strömstad, Sweden ( ∗ author for correspondence) Key words: protocols, RAPD, Littorina saxatilis, L. obtusata, L. fabalis, L. littorea, extracting DNA, amplifying DNA Abstract Randomly amplified polymorphic DNA (RAPD) is a fast and useful method of genome marking that is useful for studies of, for example, parentage, mating patterns, taxonomy of sibling species and intra-specific population genetic structures. Here we compare three different procedures for extracting high molecular weight genomic DNA; phenol-chloroform, hexadecyltrimethyl ammonium-bromide (CTAB) and Chelex 100. Double phenol-chloroform and CTAB extractions both generated high amounts of high quality DNA while Chelex 100 failed to do so. We also compared PCR-amplification with different concentrations of template DNA and found that 1–2 ng per 25 μl of amplification cocktail gave the best results. Amplifying DNA prepared by the three extraction methods revealed that DNA extracted with double phenol-chloroform gave the clearest bands. The double phenol-chloroform extraction seems thus the most suitable extraction method for RAPD in Littorina, however Chelex may be the only method useful for extracting DNA from very small individuals, for example, pre-hatching stages. Introduction Allozyme markers have successfully been used in studies of littorinid gastropods for more than 15 years, particularly in the fields of taxonomy (e.g. Ward & Warwick, 1980; Mastro et al., 1982; Janson, 1985; Johannesson & Johannesson, 1990) and evolution- ary ecology (Janson, 1987; Johannesson et al., 1995; Rolán-Alvarez et al., 1995). Recently, new molecular techniques have become available and there is already an increasing emphasis on the use of DNA character- istics as markers in taxonomic studies (Crossland et al., 1993; Rumbak et al., 1994; Crossland et al., 1996; Reid et al., 1996). Furthermore, it may be expected that studies of gene flow, hybridisation, parentage and mating patterns in littorinid species may soon profit from the use of DNA-markers as in a number of other species (Avise, 1994; Schierwater et al., 1994). Rum- bak et al. (1994) and Reid et al. (1996) successfully used mitochondrial cytochrome-b, 12S and 16S ribo- somal RNA sequences to reconstruct the phylogeny of the genus Littorina. Likewise mtDNA has been used for identification of sibling species of blue mus- sels, Mytilus (Geller & Powers, 1994; Heath et al., 1996). In these cases PCR (polymerase chain reaction) and widely applicable universal primers were used to amplify regions of the mtDNA genes. For example, fragments of the 12S and 16S ribosomal RNA genes and of the cytochrome-b gene, were amplified and se- quenced in the phylogenetic analysis of the species of Littorina (Rumbak et al., 1994; Reid et al., 1996). An alternative to sequence amplified genes and a much simpler method is RAPD (random ampli- fied polymorphic DNA) (Welch & McClelland, 1990; Williams et al., 1990). This technique also relies on PCR to amplify arbitrary sequences of DNA using short synthetic oligonucleotide primers. The amplified products are, however, directly used as anonymous ge- netic markers. RAPD has successfully been applied in a number of taxonomic and population genetic studies, for example, in marine mussels (Adamkewicz & Ha- rasewych, 1994; Patwary et al., 1994; Tyler-Walters &