Molecular Ecology Notes (2001) 1, 103–105 © 2001 Blackwell Science Ltd Blackwell Science Ltd TECHNICAL NOTE Technique for analysis of bryozoan mitochondrial DNA J. S. PORTER, D. O. F. SKIBINSKI, J. LEAMON and P. J. HAYWARD School of Biological Sciences, University of Wales, Swansea, Singleton Park, Swansea, SA2 8PP, UK Abstract The use of non-specific mitochondrial DNA (mtDNA) primers can frequently result in the erroneous amplification of DNA from contaminating organisms or from mtDNA nuclear inserts. A reliable method for deriving species-specific mtDNA polymerase chain reaction (PCR) primers using genomic and mtDNA enriched preparations from the same individual is described. Keywords: Alcyonidium diaphanum, Bryozoa, mitochondria, mtDNA, PCR Received 14 September 2000; revision accepted 16 November 2000 Polymerase chain reaction (PCR) analysis of mitochondrial DNA (mtDNA) from a relatively little studied group such as bryozoa is hampered by lack of prior sequence data and by the small amount of tissue available in many species. This hinders design of species-specific PCR primers. The task is made more difficult by the presence of bacteria and phytoplankton on colony surfaces and in the gut. Use of nonspecific mtDNA primers with genomic DNA preparations might result in amplification of foreign DNA or bryozoan nuclear DNA inserts. To date, universal mtDNA primers have been used in bryozoa. Dick et al. (2000) amplified, cloned and sequenced a 16S rRNA gene segment and constructed phylogenetic trees of 23 Gymnolaemate bryozoan species; Schwaninger (1999) sequenced cytochrome oxidase I (COI) PCR products to study population differentiation in the marine bryozoan Membranipora. Our testing of universal primers for COI and for the 12S and 16S rRNA genes gave multibanded PCR profiles with genomic DNA from the bryozoan Alcyonidium diaphanum (Hudson 1762), and raised concern about primer specificity. Nuclear copies of mtDNA sequences occur in many spe- cies and, although interesting, may confound phylogenetic analyses (see Zhang & Hewitt 1996). Nuclear insertions can occur repeatedly during evolution and be highly or little diverged from normal mtDNA (e.g. Bensasson et al. 2000). Thus, phylogenetic affinity cannot be relied upon to dis- tinguish mtDNA from nuclear pseudogenes. Nuclear contamination can frustrate interpretation in several ways. In monkeys, mtDNA primers prepared from related species gave PCR bands containing multiple nuclear DNA as well as mtDNA sequences even with mtDNA purified by caesium chloride centrifugation (Collura & Stewart 1995). Differences in the relative amounts of nuclear DNA and mtDNA between elephant blood and hair caused differential amplification of the two genomes (Greenwood & Paabo 1999). Apparent mtDNA heteroplasmy in humans was subsequently attributed to coamplification of nuclear mtDNA pseudogenes (Wallace et al. 1997). Although appar- ent frameshift or nonsense mutations are often present in mtDNA like nuclear sequences, protein-coding potential has also been observed (Collura et al. 1996). The use of mtDNA enriched preparations for PCR ana- lysis has been advocated previously (e.g. Burgener & Hubner 1998). However, this cannot solve all the above problems when nonspecific primers alone are available. Because it is easy to extract genomic DNA and apply such primers, pitfalls can occur easily. We describe here a tech- nique exploiting the high concentration of mtDNA in oocytes. A mtDNA extraction procedure is applied to oocyte rich tissue: a conventional genomic DNA extrac- tion procedure to somatic tissue. The former procedure enriches for mtDNA generally compared with the latter, whether this mtDNA be bryozoan or from a contaminating organism. In addition, the use of oocyte rich tissue enriches for mtDNA that is specifically bryozoan, compared with the use of somatic tissue. PCR with universal primers gives distinctively different banding patterns for the two pre- parations. Bands specific to the mtDNA preparation are likely to derive from mtDNA rather than nuclear DNA. mtDNA was extracted from the bryozoan A. diaphanum using the method of Fisher & Skibinski (1990). Tissue rich in oocytes was homogenized in an equal volume of Correspondence: D. O. F. Skibinski. Fax: 01792 295447; E-mail: d.o.f.skibinski@swansea.ac.uk