title: Multiplex genotyping based on the melting temperature of a single locked nucleic acid probe.
abstract: Locked nucleic acid (LNA) is a modified RNA nucleotide that can be incorporated at specific positions to generate probes with the desired length, melting temperature (TM), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the TM of a single dual-labeled LNA probe. Using this method, two varieties of the hairtail fish Trichiurus lepturus can be distinguished from each other, as well as from Trichiurus japonicus, based on a 1- to 2-bp difference in a fragment of mitochondrial cytochrome oxidase subunit 1. The shift in TM was 15 °C for a 1-bp mismatch and 27 °C for a 2-bp mismatch, indicating remarkable specificity. We anticipate that the method will be widely useful in applications such as species identification that require accurate, multiplex, and efficient detection of DNA polymorphisms.
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea.
authors.forename: Jeong Jin
authors.initials: JJ
authors.lastname: Ahn
authors.affiliation: Bio-Core Co., Guro-gu, Seoul 152-766, Republic of Korea.
authors.forename: Ji-Hoon
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea.
authors.forename: Youngjoo
authors.initials: Y
authors.lastname: Kim
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Hong
authors.affiliation: Department of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea.
authors.forename: Gi Won
authors.initials: GW
authors.lastname: Kim
authors.affiliation: Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea; Bio-Core Co., Guro-gu, Seoul 152-766, Republic of Korea; Department of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea. Electronic address: syhwang@hanyang.ac.kr.
authors.forename: Seung Yong
authors.initials: SY
authors.lastname: Hwang
mesh_terms: D000818:Animals; D004247:DNA / Q000032:analysis*; D003576:Electron Transport Complex IV / Q000235:genetics; D005838:Genotype; D008928:Mitochondria / Q000201:enzymology; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010473:Perciformes / Q000235:genetics; D011110:Polymorphism, Genetic; D060888:Real-Time Polymerase Chain Reaction; D044366:Transition Temperature
chemical_list: D009841:Oligonucleotides; C477371:locked nucleic acid; D004247:DNA; D003576:Electron Transport Complex IV
keywords: Genotyping; Locked nucleic acid; Melting temperature; Real-time PCR