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 (T-M), and specificity. Here, we describe a method of multiplex genotyping based on dramatic shifts in the T-M 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 T-M was 15 degrees C for a 1-bp mismatch and 27 degrees 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. (C) 2015 Elsevier Inc. All rights reserved.