Single-strand conformation polymorphism for p53 mutation by a combination of neutral pH buffer and temperature gradient in capillary electrophoresis

Cecilia Gelfi, Agnese Vigano, Sara De Palma, Pier Giorgio Righetti, Sabin Carla Righetti, Elisabetta Corna, Franco Zunino

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Abstract

A large number of point mutations in the p53 gene have been detected by capillary zone electrophoresis via single-strand conformation polymorphism (SSCP) analysis. A much improved detection sensitivity was obtained via the following modifications in running conditions: use of low-viscosity 3% hydroxyethylcellulose (HEC), a neutral pH (pH 6.8) buffer, in which the standard Tris moiety was substituted with a 2-(N-morpholino) ethanesulfonic acid (MES)/Tris mixture, use of SYBR Green II for improved fluorescent signal at the lower pH adopted; and, finally, the use of a temperature gradient in the 15-25°C interval, for favoring the conformational transitions in the mutated samples. The typical temperature gradient activated had a slope of 2°C/min and were induced externally. A total of 24 samples from affected patients, both in the homo and heterozygous state, were analyzed. All the mutations could be detected by this improved protocol, raising the sensitivity from the standard ca. 80% of conventional SSCP to essentially 100% with the present methodology. All the mutations were confirmed by sequence analysis of the affected samples.

Original languageEnglish
Pages (from-to)1517-1523
Number of pages7
JournalElectrophoresis
Volume23
Issue number10
DOIs
Publication statusPublished - 2002

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Keywords

  • p53
  • Point mutations
  • Single-strand conformation polymorphism

ASJC Scopus subject areas

  • Clinical Biochemistry

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