Abstract
We introduce a method for the detection of single-nucleotide polymorphisms (SNPs) by polyacrylamide gel electrophoresis (PAGE) with an additive Zn2+–cyclen complex (cyclen is 1,4,7,10-tetraazacyclododecane), called “Zn2+–cyclen–PAGE.” The method is based on the difference in mobility of mutant DNA (of 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 polymerase chain reaction based heteroduplexing technique, the method is more accurate than many other conventional PAGE-based methods, as shown by clear gel-shifting bands because of the separation of heteroduplex and homoduplex DNAs. We demonstrate SNP mapping and heterozygosity screening in a human cardiac sodium channel gene (i.e., SCN5A) that relates to inherited arrhythmia syndromes using Zn2+–cyclen–PAGE.
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Acknowledgements
The authors would like to express their sincere gratitude to the subjects who provided blood samples for this study. We gratefully acknowledge helpful discussions with Mitsuhiko Shionoya (University of Tokyo), Kazuaki Chayama (Hiroshima University), and Yukiko Nakano (Hiroshima University). We also wish to thank the Research Center for Molecular Medicine and the Analysis Center of Life Science, Hiroshima University, Japan, for the use of their facilities. This work was supported by Grants-in-Aid for Scientific Research (B) (19390011) and (C) (19590040) from the Japan Society of the Promotion of Science (JSPS), a Grant-in-Aid for Young Scientists (B) (18790120) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT), a research grant for Promoting Technological Seeds from the Japan Science and Technology Agency (JST), and research grants from the Ube Foundation (The Watanabe Memorial Award), the Iketani Science and Technology Foundation, and the Takeda Science Foundation.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC 2003
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Kinoshita-Kikuta, E., Kinoshita, E., Koike, T. (2009). Zn(II)–Cyclen Polyacrylamide Gel Electrophoresis for SNP Detection. In: Komar, A. (eds) Single Nucleotide Polymorphisms. Methods in Molecular Biology™, vol 578. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-411-1_10
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DOI: https://doi.org/10.1007/978-1-60327-411-1_10
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