Universal fluorescent labeling of amplification products using locked nucleic acids

Amplification/hybridization‐based genetic analyses using primers containing locked nucleic acids ( LNA s) present many benefits. Here, we developed a novel design for universal fluorescent PCR using LNA s. Universal fluorescent PCR generates intermediate nonlabeled fragments and final fluorescent fragments in a two‐step amplification process that uses locus‐specific primers with universal tails and universal fluorescent primers. In this study, a few standard nucleotides were replaced with LNA s only in the fluorescent universal primers. The sequence of the fluorescent universal primer significantly affected the amplification efficiency. For primers with three LNA s, the fluorescent primers with stable M 13(‐47) sequences provided the most efficient signal (approximately tenfold higher than the primers with M 13(‐21) sequences at lower T m values). Moreover, AT ‐rich LNA substitutions in the fluorescent primers produced much lower amplification efficiencies than GC ‐rich substitutions. GC ‐rich LNA s produced greater differences in T m values among primers, and resulted in the preferential production of fluorescently labeled amplicons. The specificity and sensitivity of LNA ‐containing fluorescent primers were assessed by genotyping eight STR s in J apanese individuals, and full STR profiles could be generated using as little as 0.25 ng of genomic DNA . The method permitted clear discrimination of alleles and represents sensitive STR genotyping at a reduced cost.