Detection of the Gua/Cyt-to-Cyt/Gua mutation in a Gua/Cyt-lined sequence using Zn2+–cyclen polyacrylamide gel electrophoresis

Kinoshita Eiji

Kinoshita-Kikuta Emiko

Yoshimoto Miki

Koike Tohru

Analytical Biochemistry

We have previously reported a method for the detection of single-nucleotide polymorphisms by polyacrylamide gel electrophoresis (PAGE) with an additive Zn2+–cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecan), called Zn2+–cyclen–PAGE. The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE, which is due to Zn2+–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 of 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 Zn2+–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.

Guanine

Cytosine

SNP

Mutation

Heteroduplex

Zn2+–cyclen

Electrophoresis

Medical sciences [ 490 ]

eng

Academic Press Inc.

Elsevier Science

Copyright (c) 2008 Elsevier Inc.

[ISSN] 0003-2697

[DOI] 10.1016/j.ab.2008.05.040

[NCID] AA00524867

[DOI] http://dx.doi.org/10.1016/j.ab.2008.05.040