Comparative Analyses of Four Different Methods of Genotyping ALDH2

Authors

  • Sakae Itoga,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Toru Nanmoku,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Takayuki Uchimoto,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Masahiko Sunaga,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Masahiko Nezu,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Takeshi Tomonaga,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Shoji Harada,

    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
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  • Fumio Nomura

    Corresponding author
    1. Department of Molecular Diagnosis (SI, TU, MS, MN, TT, FN), Graduate School of Medicine, Chiba University, Chiba, Japan; Department of Clinical Laboratory Medicine (TN), Tsukuba University Hospital, Tsukuba, Japan; and Department of Biochemistry and Integrative Medical Biology School of Medicine (SH), Keio University, Tokyo, Japan.
      Reprint requests: Fumio Nomura, MD, Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan; Fax: 043-226-2367; E-mail: fnomura@ho.chiba-u.ac.jp.
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Reprint requests: Fumio Nomura, MD, Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, 1-8-1, Inohana, Chuo-ku, Chiba-shi, Chiba 260-8670, Japan; Fax: 043-226-2367; E-mail: fnomura@ho.chiba-u.ac.jp.

Abstract

Background: A number of methods of genotyping single nucleotide polymorphisms (SNPs) are currently available, ranging from the traditional restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) to more sophisticated technologies. We determined the utility of three novel methods by genotyping aldehyde dehydrogenase-2 (ALDH2).

Methods: DNA was isolated from blood samples of 241 control subjects and 74 patients with esophageal cancer. The utility of three novel genotyping methods—melting curve analysis using a LightCycler, SNaPshot analysis using an ABI PRISM 310 genetic analyzer, and denaturing high-performance liquid chromatography using a WAVE DNA fragment analysis system—were compared with that of conventional fluorescent-based polymerase chain reaction (PCR)-SSCP using an ALF express DNA sequencer.

Results: The frequency of the mutant ALDH2*2 allele was significantly higher in patients with esophageal cancer (27.7%) than in control subjects (16.2%; p < 0.01; habitual alcohol drinkers). The melting curve analysis was accurate, more rapid, and easier to use than the SNaPshot analysis or denaturing high-performance liquid chromatography analysis. Fluorescent-based PCR-SSCP proved useful for analyzing a large number of samples.

Conclusion: Melting curve analysis using the LightCycler is suitable for the genotyping of small numbers of samples in a routine clinical setting; fluorescent-based PCR-SSCP analysis using the ALF express DNA sequencer can be used for large-scale genotyping in epidemiologic studies.

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