Multiplex genotyping based on the melting temperature of a single locked nucleic acid probe |
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Authors: | Jeong Jin Ahn Ji-Hoon Kim Youngjoo Kim Ji Young Hong Gi Won Kim Seung Yong Hwang |
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Affiliation: | 1. Department of Bio-Nanotechnology, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea;2. Bio-Core Co., Guro-gu, Seoul 152-766, Republic of Korea;3. Department of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea |
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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. |
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Keywords: | Locked nucleic acid Genotyping Real-time PCR Melting temperature |
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