Polymerase Chain Reaction-Direct Sequencing Devised for Mutation Analysis of β-globin Gene : Molecular Diagnosis of Sickle Cell Anemia and Unstable Hemoglobin Burke

  • Ohnuki Yuko
    Department of Clinical Pathology, Kitasato University School of Medicine
  • Wada Chieki
    Department of Clinical Pathology, Kitasato University School of Medicine
  • Toyo-oka Yuko
    Laboratory of Virology and DNA Research, Kitasato University Hospital
  • Kamata Kouju
    Department of Internal Medicine, Kitasato University School of Medicine
  • Okubo Michihito
    Department of Internal Medicine, Sano Kousei Sougou Hospital
  • Suzuki Haruo
    Department of Biophysical Chemistry, Kitasato University School of Medicine
  • Ohtani Hideki
    Department of Clinical Pathology, Kitasato University School of Medicine

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Other Title
  • PCRダイレクトシークエンス法を用いたβ-グロビン遺伝子変異の解析 : 遺伝子診断が可能であった鎌状赤血球性貧血症および不安定ヘモグロビンBurke

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Polymerase chain reaction (PCR), a technique allowing enzymatic amplification of specific genes in vitro, was used to devise a method of analysing β-globin transcripts. To evaluate the method for clinical reliability, we used it to investigate mutations of the β-globin transcripts in blood samples derived from two patients having abnormal hemoglobin. Employing PCR-direct sequencing, this procedure entailed the isolation of total cellular RNA from peripheral blood, then the β-globin transcripts were amplified by reverse transcriptase-PCR. A second amplification was performed with the primer possessing the d (T)_<15> tail, and single-strand PCR products were purified with Dynabeads-oligo (dT)_<25>. Cycle sequencing reaction was then conducted from a singlestrand DNA template. Our modified version of PCR-direct sequencing enabled sufficient sequencing of the β-globin transcripts and the detection of sickle cell anemia and unstable hemoglobin Burke by molecular diagnosis. From a very minimal amount of clinical specimen, PCR-direct sequencing was shown to provide rapid and sensitive analysis of mutations of genes and transcriptions associated with the diseases investigated. This approach is deemed a reliable and useful diagnostic modality with high potential for extended clinical application.

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