Zn(II)-cyclen polyacrylamide gel electrophoresis for SNP detection.
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.
Kinoshita-Kikuta
Emiko
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan.
Kinoshita
Eiji
E
Koike
Tohru
T
D000080027:Cyclams; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005091:Exons / Q000235:genetics; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D015222:Sodium Channels / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D000080027:Cyclams; D006571:Heterocyclic Compounds; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009692:Nucleic Acid Heteroduplexes; D022821:RNA Splice Sites; C568320:SCN5A protein, human; D015222:Sodium Channels; C038072:cyclen; D015032:Zinc