Temperature-programmed capillary electrophoresis for the analysis of high-melting point mutants in thalassemias

Cecilia Gelfi, Pier Giorgio Righetti, Maurizio Travi, Silvia Fattore

Research output: Contribution to journalArticlepeer-review

Abstract

The behavior of different sieving polymers for unambiguous determination of point mutations in genomic DNA, based on electrophoresis in thin capillaries, is evaluated. High melters from thalassemia patients are separated by exploiting the principle of denaturing gradient gel electrophoresis, in fact, of its variant utilizing temperature gradients (TGGE), along the migration path, encompassing the melting points of both homo- and heteroduplex, polymerase chain reaction (PCR)-amplified DNA fragments. Unlike TGGE, where the temperature gradient exists along the separation space, the denaturing temperature gradient in the fused-silica capillaries is time-programmed, so as to reach the T(m)'s of all species under analysis prior to electrophoretic transport past the detector window. The DNA fragments are injected in a capillary maintained (by combined chemical and thermal means) just below the expected T(m) values. The ΔT applied is rather minute (1-1.5°C) and the temperature gradient quite shallow (e.g., 0.05°C/min). The denaturing thermal gradient is generated internally, via Joule heat produced by voltage ramps. This method is applied to the analysis of the most common point mutations in thalassemias, characterized by being high melters (in the temperature range of 60-62°C) in presence of 6 M urea. Point mutants are fully resolved into a spectrum of four bands only when poly(N-acryloylaminopropanol) and hydroxyethylcellulose are used. However, the former offers the best separation capability at such high temperatures.

Original languageEnglish
Pages (from-to)724-731
Number of pages8
JournalElectrophoresis
Volume18
Issue number5
DOIs
Publication statusPublished - May 1997

Keywords

  • Capillary electrophoresis
  • DNA electrophoresis
  • N-Subtituted acrylamides
  • Thalassemias

ASJC Scopus subject areas

  • Clinical Biochemistry

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