Rapid and practical detection of β-globin mutation causing β-thalassemia by fluorescence-based PCR-single-stranded conformation polymorphism analysis

Asako Takahashi-Fujii, Yoshizumi Ishino, Ikunoshin Kato, Yasuyuki Fukumaki

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

We report a useful method for daily clinical examination for the diagnosis of thalassemia. We applied a fluorescence-based image analyser to a non-radioisotopic PCR-single-stranded conformation polymorphism (SSCP) analysis to detect mutations in the β-globin gene. PCR primers were labelled with rhodamine X and the amplified fragments from the β-globin gene were resolved by non-denaturing polyacrylamide gel electrophoresis. After loading, the glass plate was set in the image analyser and scanned with a green laser. We detected four common mutations in exon I and two major mutations in intron 1 of the β-globin gene isolated from patients with β-thalassemia. Moreover, to discriminate mutations and natural polymorphisms, we used primers including one base mismatch at the polymorphic site, which can substitute the polymorphic site by a constant base in the PCR amplified fragment. This fluorescence-based system was simple to operate and results were obtained rapidly as clear image data. Therefore, once the optimal conditions of the electrophoresis are determined, this system will be suitable for daily clinical use, especially for screening of molecular defects and for the prenatal diagnosis of genetic disorders.

Original languageEnglish
Pages (from-to)385-393
Number of pages9
JournalMolecular and Cellular Probes
Volume8
Issue number5
DOIs
Publication statusPublished - Oct 1 1994

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Thalassemia
Globins
Fluorescence
Polymerase Chain Reaction
Mutation
Genes
Native Polyacrylamide Gel Electrophoresis
Inborn Genetic Diseases
Rhodamines
Prenatal Diagnosis
Introns
Glass
Electrophoresis
Exons
Lasers

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

Cite this

Rapid and practical detection of β-globin mutation causing β-thalassemia by fluorescence-based PCR-single-stranded conformation polymorphism analysis. / Takahashi-Fujii, Asako; Ishino, Yoshizumi; Kato, Ikunoshin; Fukumaki, Yasuyuki.

In: Molecular and Cellular Probes, Vol. 8, No. 5, 01.10.1994, p. 385-393.

Research output: Contribution to journalArticle

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