Application of denaturing gradient gel electrophoresis (DGGE) to screen for mutations of the human glucocorticoid receptor alpha gene (hGRalpha).
OBJECTIVE
In a previous publication, we had presented a sensitive method to detect mutations of the segment of the human glucocorticoid receptor alpha (hGRalpha) gene encoding the ligand binding domain (LBD) and part of the DNA binding domain (DBD) of hGRalpha, as several types of glucocorticoid resistance syndromes have been correlated with mutations in the respective nucleotide sequences. However, mutations affecting various regions covering the whole length of hGRalpha are increasingly reported in a variety of disease states. We now present an expanded screening methodology to detect mutations covering the whole length of hGRalpha.


METHODS
We developed a sensitive, simple screening PCR-DGGE method to detect mutations in the aminoterminal domain and DNA-binding domain of the hGRalpha. Wild type hGRalpha cDNA and mutant samples were included in the analysis to ensure the accuracy and sensitivity of the method.


RESULTS
The PCR-DGGE method identified the mutant samples and discriminated them from wild type hGRalpha.


CONCLUSIONS
The method described is accurate, sensitive, simple, cheap and fulfills the critera for a screening method which will be useful in delineating possible involvement of hGRalpha mutations in the aetiopathology of diseases correlated to derangements of glucocorticoid action.
Tsolakidou
Amalia F
AF
Department of Biological Chemistry, Medical School, National University of Athens, 75 M Asias str, Goudi, 115 27 Athens, Greece.
Coulocheri
Stavroula A
SA
Sekeris
Constantine E
CE
Moutsatsou
Paraskevi
P
D004252:DNA Mutational Analysis / Q000379:methods; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011965:Receptors, Glucocorticoid / Q000235:genetics*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D011965:Receptors, Glucocorticoid; C411470:glucocorticoid receptor alpha