Allelotyping large numbers of samples by allele-specific polymerase chain reaction (PCR) can be problematic if the DNA samples to be tested are of highly variable concentration. On the one hand, analysis of dilute DNA samples often requires nested PCR to produce a product of sufficient yield to be detectable on ethidium bromide-stained agarose gels. Such two-step assays require additional reagents, are labor-intensive, and have a higher risk of contamination. On the other hand, the specificity of allele-specific PCR assays can be lost at high input DNA concentrations. Large population-based genetic studies using DNA from varied sources would benefit from one-tube assays that could detect mutations in samples over a wide range of concentration. We describe a one-tube nested allele-specific PCR-based assay, in which the input DNA concentration has little effect on the assay's yield or specificity. An assay using this method is highly sensitive and specific, and was used to type several thousand DNA samples, obtained from various sources, for a G to A transition at human transthyretin codon 122. Similar assays could be readily adapted to any high-throughput allelotype assay where input DNA is of highly variable concentration.