Single-strand conformation polymorphism analysis by capillary zone electrophoresis in neutral pH buffer

Cecilia Gelfi, Agnese Viganó, Mario Curcio, Pier Giorgio Righetti, Sabina Carla Righetti, Elisabetta Corna, Franco Zunino

Research output: Contribution to journalArticle

Abstract

Sensitivity of single-strand conformation polymorphism (SSCP) analysis of polymerase chain reaction (PCR) products was reported to be lower in capillary zone electrophoresis (CZE) compared to conventional slab gel electrophoresis. We examined the effects of buffer ion type, pH, and temperature in an attempt to improve the mutation detectability in the SSCP- CZE mode. It was noted that, by utilizing short-chain polyacrylamide as sieving media while simultaneously lowering the temperature, there was no improvement of conformer detectability. On the contrary, there was a large increment in conformers' resolution by running samples in a lower-pH buffer system. The effects of different buffering ions and pH values were investigated. By using a new buffer system, consisting of 35 mM 2-(N- morpholino)propanesulfonic acid (MES), 30 mM tris(hydroxymethyl)aminomethane (Tris), 1 mM ethylene diaminetetraacetic acid (EDTA), pH 6.8, and keeping constant all the other conditions, such as temperature, sieving, applied voltage, capillary length, and inner diameter (ID), there was a remarkable improvement in resolution and the sensitivity became comparable to that of slab gel systems.

Original languageEnglish
Pages (from-to)785-791
Number of pages7
JournalElectrophoresis
Volume21
Issue number4
DOIs
Publication statusPublished - 2000

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Keywords

  • p53
  • Point mutation
  • Single-strand conformation polymorphism

ASJC Scopus subject areas

  • Clinical Biochemistry

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