We previously reported a method for the detection of single-nucleotide polymorphisms by polyacrylamide gel electrophoresis (PAGE) with an additive Zn(2+)-cyclen complex (cyclen=1,4,7,10-tetraazacyclododecane), called Zn(2+)-cyclen-PAGE. The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE that is due to Zn(2+)-cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. In combination with a heteroduplexing technique, the method is more accurate, as shown by clear gel-shifting bands. However, the question remains as to whether the Gua/Cyt-to-Cyt/Gua mutation, which is far apart from the Thy/Ade base (i.e., in a Gua/Cyt-lined sequence), can be detected by this Thy-dependent method. In this study, we determined the potency of Zn(2+)-cyclen-PAGE for the detection of the Gua/Cyt-to-Cyt/Gua single substitutions in some artificial Gua/Cyt-lined sequences derived from a human cardiac sodium channel gene, SCN5A. All Gua/Cyt-to-Cyt/Gua substitutions in the 28-set samples tested, which are 1 to 10 bases away from the nearest Thy/Ade, were successfully detected by designing DNA fragments of the appropriate length.