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Nucleic Acids Research, 2002, Vol. 30, No. 22 e126
© 2002 Oxford University Press

A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+–cyclen complex

Emiko Kinoshita-Kikuta, Eiji Kinoshita1 and Tohru Koike*

Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences and 1 Department of Cardiovascular Physiology and Medicine, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan

*To whom correspondence should be addressed. Tel: +81 82 257 5323; Fax: +81 82 257 5336; Email: tkoike{at}hiroshima-u.ac.jp

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn2+– cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). 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. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn2+–cyclen–PAGE).


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E. Kinoshita, E. Kinoshita-Kikuta, H. Kojima, Y. Nakano, K. Chayama, and T. Koike
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