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Universal fluorescent labeling of amplification products using locked nucleic acids
Author(s) -
Asari Masaru,
Oka Kumiko,
Omura Tomohiro,
Maseda Chikatoshi,
Tasaki Yoshikazu,
Shiono Hiroshi,
Matsubara Kazuo,
Matsuda Mitsuyoshi,
Shimizu Keiko
Publication year - 2013
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201200215
Subject(s) - amplicon , fluorescence , locked nucleic acid , primer (cosmetics) , genotyping , nucleic acid , microbiology and biotechnology , primer dimer , biology , locus (genetics) , dna , genomic dna , polymerase chain reaction , genetics , oligonucleotide , chemistry , genotype , gene , multiplex polymerase chain reaction , physics , organic chemistry , quantum mechanics
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.