Sequencing will be performed using PRISM Ready Reaction Dye Terminator Cycle Sequencing kits (Applied Biosystems), following protocols suggested by the manufacturer. Unincorporated nucleotides and primers are removed from sequencing reactions using Centri-Sep columns (Princeton Separations), and the cleaned sequencing reactions are ethanol precipitated, re-suspended in formamide-EDTA and electrophoresed in 6% Sequagel (National Diagnostics) acrylamide gels on either the Applied Biosystems 370A or 373A automated fluorescent DNA sequencer (see "Facilities, Equipment and Other Resources"). In general, following a 10-hour run we can read up to 400 bases, and two runs are possible in a twenty-four hour period on each machine. Sequence and gel image files are exported via Ethernet from the DNA sequencer to computers in the Donoghue lab for proofreading and sequence editing (SeqEd ver. 1.0.1, Applied Biosystems), and these files are routinely archived on 128 MB optical discs. Applied Biosystems software has been used for automated base calling. Both strands will be sequenced as a means of eliminating artifacts and ambiguities. Multiple sequence alignments will be facilitated using Clustal V (Higgins et al., 1992) and Malign (Wheeler and Gladstein, 1993).
In light of our previous experience, we anticipate little difficulty in obtaining sequences from the DNA regions of interest. Mitochondrial rDNA may yield ca 600-850 bp, nuclear rDNA ca. 1.2 kilobases, and ITS ca. 600 bp. Despite high copy number (e.g., of the nuclear ITS region) it appears in many cases that sequences are effectively homogenized, which allows direct sequencing of pooled PCR products. However, divergent paralogues have been discovered in some cases (e.g., Ritland et al., 1993; Suh et al., 1993) and may be common in some groups (Baldwin et al., 1995). The presence of different repeat types has in some cases been discovered through direct sequencing of PCR products, i.e., by observing more than one band/peak per site, and cloning is required for accurate characterization of such variation (Baldwin et al., 1995).