D. Bitenc et J. Marc, Optimization of single-stranded conformation polymorphism (SSCP) analysis for screening for the estrogen receptor-alpha gene polymorphism P325P, CLIN CH L M, 39(7), 2001, pp. 612-614
Since there are no theoretical models for single-stranded conformation poly
morphism (SSCP) analysis, conditions for detecting specific mutation must b
e found experimentally. Previously, a time-consuming (22 hours) SSCP method
was used for the detection of polymorphism in codon 325 (CCC to CCG; P325P
) in exon 4 of estrogen receptor-alpha gene. The aim of our work was to stu
dy different gel loading buffers, additives to polyacrylamide gel, voltages
, running times and temperatures of electrophoresis, in order to develop a
better and faster SSCP analysis for screening of P325P polymorphism. Our re
sults show that a low ionic strength gel loading buffer and 10% addition of
glycerol to the 8% polyacrylamide gel (37:1) are essential for the good se
paration of mutated and wild-type single stranded conformers of exon 4. The
most suitable conditions for electrophoresis were 300 V, 5 h and 22 degree
sC. We concluded that a much faster SSCP analysis for sreening of P325P pol
ymorphism of estrogen receptor-alpha gene was developed. Although our final
result could be applied only to the detection of the described genetic pol
ymorphism, we hope that the results of our study will be helpful to analyst
s using SSCP analysis in their mutation screening programs.