Conditions for single strand conformation polymorphism (SSCP)analysis with broad applicability: a study on the effects of acrylamide, buffer and glycerol concentrations in SSCP analysis of exons of the p53 gene).
We examined the influence of electrophoretic conditions on the detectability of small sequence alterations in DNA fragments by single strand conformation polymorphism (SSCP) analysis. Three acrylamide concentrations, 7.5, 14 and 20%, were selected, and all three gel types were prepared with four different Tris/borate/EDTA buffer concentrations. In addition, all these twelve gels were prepared both with and without 10% glycerol. All electrophoretic runs were performed at ambient temperature of 20-24 degrees C. The resulting 24 different electrophoretic conditions were used for the SSCP analysis of DNA fragments of exons 5, 7 and 8 of the human p53 gene; the results were evaluated primarily in relation to detectability of mutants. Six out of the 24 conditions permitted the detection of all mutants. For practical reasons, 14% acrylamide, 44.5 mmol/1 Tris, 44.5 mmol/1 boric acid, 1 mmol/1 EDTA, pH 8.0, with and without addition of glycerol was chosen as the most suitable. These "selected conditions" were applied in the SSCP analysis of an arbitrarily chosen set of mutant DNA fragments with single base exchanges,and all but one of seven mutants were detected in the gel system containing glycerol. Our results indicate that the set of "selected conditions" is of broad applicability, permitting the detection of even small sequence differences like single base exchanges with high reliability. It should prove especially useful in screening for unknown mutations.
Teschauer
W
W
Abteilung fur Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Munchen, Germany.
Mussack
T
T
Braun
A
A
Waldner
H
H
Fink
E
E
D000178:Acrylamides; D002021:Buffers; D004247:DNA / Q000032:analysis* / Q000235:genetics; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics; D004586:Electrophoresis; D005091:Exons; D016158:Genes, p53; D005990:Glycerol; D006801:Humans; D007700:Kinetics; D009154:Mutation; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature
D000178:Acrylamides; D002021:Buffers; D004277:DNA, Single-Stranded; D004247:DNA; D005990:Glycerol