Premium
Detection of p53 point mutations by double‐gradient, denaturing gradient gel electrophoresis
Author(s) -
Gelfi Cecilia,
Righetti Sabina C.,
Zunino Franco,
Della Torre Gabriella,
Pierotti Marco A.,
Righetti Pier Giorgio
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181533
Subject(s) - temperature gradient gel electrophoresis , single strand conformation polymorphism , microbiology and biotechnology , point mutation , biology , gel electrophoresis , gene , chromatography , chemistry , exon , mutation , genetics , 16s ribosomal rna
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.