Teschauer W, Mussack T, Braun A, Waldner H, Fink E
Abteilung fur Klinische Chemie und Klinische Biochemie in der Chirurgischen Klinik und Poliklinik, Munchen, Germany.
Eur J Clin Chem Clin Biochem. 1996 Feb;34(2):125-31. doi: 10.1515/cclm.1996.34.2.125.
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.
我们通过单链构象多态性（SSCP）分析研究了电泳条件对DNA片段中小序列改变检测能力的影响。选择了三种丙烯酰胺浓度，即7.5%、14%和20%，并使用四种不同的Tris/硼酸/EDTA缓冲液浓度制备了所有三种类型的凝胶。此外，所有这十二种凝胶均在有和没有10%甘油的情况下制备。所有电泳运行均在20 - 24摄氏度的环境温度下进行。将所得的24种不同电泳条件用于人p53基因外显子5、7和8的DNA片段的SSCP分析；主要根据突变体的检测能力对结果进行评估。24种条件中有六种能够检测到所有突变体。出于实际原因，选择14%丙烯酰胺、44.5 mmol/L Tris、44.5 mmol/L硼酸、1 mmol/L EDTA、pH 8.0，添加和不添加甘油的条件作为最合适的条件。这些“选定条件”应用于一组任意选择的具有单碱基交换的突变DNA片段的SSCP分析，在含有甘油的凝胶系统中检测到了七个突变体中的除一个之外的所有突变体。我们的结果表明，“选定条件”集具有广泛的适用性，能够以高可靠性检测甚至像单碱基交换这样的小序列差异。它在筛选未知突变方面应该特别有用。
