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

Kohei Maruyama, Haruko Takeyame, Etsuo Nemoto, Tsuyoshi Tanaka, Kiyoshi Yoda, Tadashi Matsunaga

Research output: Contribution to journalArticle

36 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)687-694
Number of pages8
JournalBiotechnology and Bioengineering
Volume87
Issue number6
DOIs
Publication statusPublished - 2004 Sep 20
Externally publishedYes

Fingerprint

Bacterial Genes
Aldehyde Dehydrogenase
Nucleotides
Polymorphism
Aldehydes
Single Nucleotide Polymorphism
Genes
Bacterial DNA
DNA
Streptavidin
Fluorescence
Biotin
Polymerase Chain Reaction
Temperature
Fluorescein-5-isothiocyanate
Homozygote
Heterozygote
Fluorescent Dyes
Particle Size
Heating

Keywords

  • Aldehyde dehydrogenase 2 gene
  • Automated system
  • Bacterial magnetic particles (BMPs)
  • DNA dissociation curve analysis
  • Single nucleotide polymorphism (SNP)

ASJC Scopus subject areas

  • Biotechnology
  • Microbiology

Cite this

Single nucleotide polymorphism detection in aldehyde dehydrogenase 2 (ALDH2) gene using bacterial magnetic particles based on dissociation curve analysis. / Maruyama, Kohei; Takeyame, Haruko; Nemoto, Etsuo; Tanaka, Tsuyoshi; Yoda, Kiyoshi; Matsunaga, Tadashi.

In: Biotechnology and Bioengineering, Vol. 87, No. 6, 20.09.2004, p. 687-694.

Research output: Contribution to journalArticle

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