24] Detection of mutations and polymorphisms of Gsα subunit gene by denaturing gradient Gel electrophoresis

Abstract

The genomic structure of the α subunit of the guanine nucleotide-binding protein G s (G s α ) is composed of 13 exons and 12 introns spanning more than 20 kilobases (kb), whereas the transcribed message of the gene is approximately 1.9 kb in length. Denaturing gradient gel electrophoresis (DGGE) analysis detected several disease-related mutations in individuals affected with Albright hereditary osteodystrophy (AHO) and with the McCune–Albright syndrome (MAS), and it also uncovered a polymorphism that was instrumental to the genetic linkage mapping of the G s α gene. This chapter reviews some basic principles of DNA denaturation and DGGE applications and the specific experimental protocols applied to the analysis of G s α . Many aspects of DGGE are also reviewed in the chapter. In DGGE experiments a temperature gradient is supplanted by a chemical gradient made of a linearly increasing concentration of a urea–formamide mixture in a polyacrylamide gel. Because the denaturation properties of the urea–formamide gradient are reasonably equivalent to those of a thermal gradient at temperatures near the melting temperatures, electrophoresis is performed at 60°.