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

Abstract

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 °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.

Temperature and pH effects on single-strand conformation polymorphism analysis by capillary electrophoresis

“…Some [Glavac and Dean, 1993;Hayashi, 1993] but not all [Ren et al, 1997;Teschauer et al, 1996] studies have demonstrated that glycerol affect the migration profile in a way that increases the sensitivity of SSCP analysis based on slab gel electro-phoresis. Recently, Kukita et al [1997] reported that the effect of glycerol is attributable to reduction of pH of the TBE buffer caused by the reaction of glycerol with borate ion.…”

Identification of the four most common β‐globin gene mutations in Greek β‐thalassemic patients and carriers by PCR‐SSCP: advantages and limitations of the method

“…Therefore, we varied the amount of DNA loaded, percentage of formamide used for denaturation, glycerol effect, methods of staining (ethidium bromide and silver nitrate), time and temperature of the electrophoretic run, power applied, and other matrixes as Mutation Detection Enhancement gel solution of Hydrolink (MDE). Such methods for optimizing the method of SSCP have been used in various studies in the literature, e.g., to detect mutations in the p53 gene (21). We also examined the reproducibility of the separation patterns obtained.…”

Single‐Stranded Conformation Polymorphism Analysis

“…. Test buffers of different composition and pH value (e.g., 0.5 Â TBE pH 8.3 or Tris-acetate pH 6.4), if necessary with and without additives such as glycerol (Teschauer et al, 1996). .…”