Detection of p53 point mutations by double-gradient, denaturing gradient gel electrophoresis

Cecilia Gelfi, Sabina C. Righetti, Franco Zunino, Gabriella Della Torre, Marco A. Pierotti, Pier Giorgio Righetti

Research output: Contribution to journalArticlepeer-review

Abstract

Genetic instability is a typical feature of tumor cells. This evidence has stimulated the development of rapid methods for detection of gene mutations. A new, improved protocol for denaturing gradient gel electrophoresis (DGGE), to screen for point mutations in genomic DNA, is reported: double gradient (DG) DGGE. In this technique, to the primary, denaturing gradient (typically 30-80% or 40-80% urea/formamide) a secondary gradient, colinear with the first, is superimposed: a porosity gradient (typically 6.5-12% polyacrylamide). The secondary gradient acts by recompacting smeared and diffuse bands of heteroduplexes, which are often indistinguishable from background fluorescence, and by augmenting the resolution between closely spaced homoduplex zones. This allows proper densitometric quantitation of the ratio of the two homoduplex bands. The reliability of this technique has been documented by detection of a number of mutations in exons 6 and 8 of the p53 gene which had escaped revelation by single-strand conformational polymorphism (SSCP) analysis. Additionally, the precise assessment of ratio of the doublet of homoduplex bands has allowed quantitation of the extent of p53 mutation in a mixed cell population extracted from a tumor specimen.

Original languageEnglish
Pages (from-to)2921-2927
Number of pages7
JournalElectrophoresis
Volume18
Issue number15
DOIs
Publication statusPublished - Dec 1997

Keywords

  • DNA
  • p53
  • Point mutations

ASJC Scopus subject areas

  • Clinical Biochemistry

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