Discovery and analysis of DNA sequence variations is an important part of genetic research (1). Denaturing high-performance liquid chromatog-raphy (DHPLC) fits the requirements in this area, addressing throughput issues by being compatible with automation while being robust, sensitive, and cost-effective. Multiplexing with fluores-cently labeled PCR products offers great hope for further improvements in sample throughput with compatible hydrophobic dye selection (2-5). However, purchasing fluorescently tagged primers for each PCR product may make its use cost-inhibitive. An added complexity is that maximum throughput involves the design of pools of four different PCR products that need to share a common melting temperature, which is not always possible.In an effort to utilize this option, we have investigated a universal fluorescent labeling (UFL) technique for its compatibility with DHPLC. This method involves a single PCR using three primers: a template-specific forward primer with a 20-bp extension at its 5′ end matching M13(-21), a template-specific reverse primer, and a fluorescently labeled M13(-21) primer (the universal primer). This technique, shown in Figure 1, involves the forward and reverse primer generating a PCR product and, when the forward primer is exhausted, the temperature is then lowered to allow the universal labeled primer to be used and to incorporate fluorescence into the accumulating PCR fragment (5). The significant advantage of this method is that the costs of project design for multiple genes or large genes are reduced considerably. Indeed, a set of only four fluorescent universal primers is required regardless of the number of PCR products to be analyzed. In addition, the same PCR product obtained from four different DNA samples that are labeled with different fluorophores can be easily pooled and run under the PCR product’s optimal conditions. This improves the flexibility in the design of the study and makes the interpretation of the results substantially easier. The first test of this technique utilized a plasmid library known as the Universal Mismatch System™ (UMS™; Genera Biosystems Pty, Ltd., Bundoora, VIC, Australia). By concen-trating on a set four plasmids that differ only in the central base with a C, G, T, or A, we were able to mix two of these plasmids at an equimolar ratio, allowing us to analyze all possible forms of single base mismatches. In this way, we can mimic naturally occurring single nucleotide polymorphisms (SNPs) in diploid genomes, making it ideal for testing the robustness of the UFL technique. This study was undertaken on the plasmid combinations that yielded the eight possible forms of mismatched bases. Illustrated in Figure 2 is one representative of the four combinations where the plasmid selection creates a CC and GG physical base mismatch. The product was analyzed by UV detection (Figure 2A). This produced four distinct peaks that represented the four species of DNA present in the sample: the two heteroduplex forms (CC and GG) followed by the two homoduplex forms (CG and GC). These same mismatches were reana-lyzed using the UFL procedure, as detailed below.Four separate PCR amplifications using one of each of the four fluores-cently labeled universal primers were performed on one UMS plasmid