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attributable to PCR inhibitors, low DNA yield in isolation, or PCR reaction issues (e.g. concentration and/or purity of chemical reagents used in the reaction, or the ability of primers to align properly on the DNA strand). The difference between number amplified and sequenced (e.g. 10 of30 dace were successfully sequenced from White River above Kenney Reservoir) is likely due to poorly amplified or poorly purified PCR product which causes the misreading of the nucleotide sequence. Samples that did not provide good sequence data were re-amplified, and sometimes DNA was re-isolated and the PCR product resubmitted. PCR products are sequenced using ABI Prism BigDye Terminator Cycle Sequencing chemistry on an ABI 377 automated sequencer. Sequence alignment and editing is performed using Sequencher 3.0 (Gene Codes Corp., Ann Arbor, MI). Phylogenetic analyses was performed using the program P AUP*, version 4.0 (Swofford, 1998). In the fifth quarter, covering the period of Apri12002-June 2002, we continued molecular work, including DNA isolation, amplification, and sequencing. The majority of the time spent on the project in this quarter was devoted to efforts in isolating DNA as well as continuing PCR and subsequent procedures. In April the two PCR machines in the lab failed and a new unit was ordered. This resulted in the loss of a month's time in PCR amplification, and thus also delayed preparation of sequencing reactions. In the beginning of May the sequencing center closed for two weeks while new technicians were trained in and new protocols were developed. It was closed for a third week because of unexpected problems with the equipment. Once the center re- opened the first several batches of DNA submitted from our lab failed to produce sequences, resulting in the loss of several weeks of work on the project. By early June the sequencing center was again producing acceptable results, although this had delayed us for over three months once the time required to re-run submitted materials was considered. , Nuclear DNA To determine which region of nuclear DNA to study, we ran PCR tests on multiple populations using primers for three different target regions. Trout DNA primers already in use in our lab did not consistently amplify DNA in speckled dace or flannelmouth sucker. After deciding on a region of the nuclear genome, based on the results of past trout studies by our lab, we used the DNA sequence databases as outlined by the National Center for Biotechnology Information. We searched for conserved DNA sequences in regions adjacent to our target region. The conserved regions were similar between various groups of animals (human v. mouse v. frog v. trout v. carp). We used this information to design primers that would amplify the desired region of DNA in the four species of fish being examined in this study. After numerous PCR experiments, we determined that the combination of one of our new primers and one of the trout primers would successfully amplify the desired region of nuclear DNA. This region is located between ribosomal subunits 5.8S and 28S and is referred to as Internal Transcribed Spacer subunit 2 (ITS2). Nuclear DNA was amplified from all specimens, but the PCR product was inconsistent, giving multiple bands, an indication of multiple sized PCR products. We attempted to resolve the amplification procedure using different temperatures, concentrations of DNA, primers, etc. That still failed to solve the problem. We even extracted individual bands from the gel, re-amplified the bands, and sequenced the resulting PCR product. None of the sequences were consistent 25