Allele‐specific, Hybridization‐based, Washing‐free Fluorescence Signal Production Platforms for Quantitation of Single‐Base Change (C → U) in RNA

Abstract

A single nucleotide polymorphism (SNP) is a single base-pair substitution that commonly constitutes the genetic variation in individuals. Because of its association with disease susceptibility and drug resistance, SNP detection is of great value in studying the variation in drug responses. Here we report four types of washing-free (i.e., separation-free or sequential addition of reagents) and polymerase chain reaction (PCR)-free fluorescence signal production platforms for quantitative detection of a single-base mismatch in RNA and compare their analytical efficiency. The RNA-templated SNP detection methods in this study are based on the allelespecific hybridization approach and/or on the allele-specific oligonucleotide ligation approach, and have been successfully applied to the quantitation of single-base mutation (C ! U) in RNA of the BCR-ABL fusion gene, by endpoint measurements of fluorescence intensity. These methods have several advantages for practical applications, such as direct discrimination of single-base mismatch of the RNA extracted from cells, no requirement of PCR amplification, performance of homogeneous detection, easy design of detection probes, and suitability for large-scale genotyping.