Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis

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Abstract
Single nucleotide polymorphism (SNP) detection for aldehyde dehydrogenase 2 (ALDH2) gene based on DNA thermal dissociation curve analysis was successfully demonstrated using an automated system with bacterial magnetic particles (BMPs) by developing a new method for avoiding light scattering caused by nanometer‐size particles when using commercially available fluorescent dyes such as FITC, Cy3, and Cy5 as labeling chromophores. Biotin‐labeled PCR products in ALDH2, two allele‐specific probes (Cy3‐labeled detection probe for ALDH2*1 and Cy5‐labeled detection probe for ALDH2*2), streptavidin‐immobilized BMPs (SA‐BMPs) were simultaneously mixed. The mixture was denatured at 70°C for 3 min, cooled slowly to 25°C, and incubated for 10 min, allowing the DNA duplex to form between Cy3‐ or Cy5‐labeled detection probes and biotin‐labeled PCR products on SA‐BMPs. Then duplex DNA‐BMP complex was heated to 58°C, a temperature determined by dissociation curve analysis and a dissociated single‐base mismatched detection probe was removed at the same temperature under precise control. Furthermore, fluorescence signal from the detection probe was liberated into the supernatant from completely matched duplex DNA‐BMP complex by heating to 80°C and measured. In the homozygote target DNA (ALDH2*1/*1 and ALDH2*2/*2), the fluorescence signals from single‐base mismatched were decreased to background level, indicating that mismatched hybridization was efficiently removed by the washing process. In the heterozygote target DNA (ALDH2*1/*2), each fluorescence signals was at a similar level. Therefore, three genotypes of SNP in ALDH2 gene were detected using the automated detection system with BMPs.

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