Screening for the β-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers

Cecilia Gelfi, Agnese Viganó, Piera Carta, Pierangela Manchia, Gian Franco Cossu, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

A novel method is reported for screening for point mutations in genomic DNA: freezone capillary electrophoresis in very acidic buffers. This method exploits the charge difference among the four different bases (C, T, A, G) in a pH window between 2.5 and 3.5, where the four titration curves fan out. The method is applied to the detection of the β-39 missense mutation in the β- globin gene in thalassemias. A 60-mer fragment straddling the mutation site has been amplified. In an isoelectric buffer (iminodiacetic acid) of pH 3.3, partial resolution between the wild type and mutated strands is obtained. In a pH 3.0 phosphate buffer, baseline resolution is achieved between the two strands in a heterozygous individual. Due to the short size of the amplified fragment, this method can only be applied to routine screening for known mutations because resolution was lost in a fragment 100 bases long.

Original languageEnglish
Pages (from-to)780-784
Number of pages5
JournalElectrophoresis
Volume21
Issue number4
DOIs
Publication statusPublished - 2000

Keywords

  • Capillary electrophoresis
  • Point mutations
  • Thalassemia

ASJC Scopus subject areas

  • Clinical Biochemistry

Fingerprint Dive into the research topics of 'Screening for the β-39 mutation in thalassemia by capillary electrophoresis in free solution in strongly acidic, isoelectric buffers'. Together they form a unique fingerprint.

Cite this