Detection of the Gua/Cyt-to-Cyt/Gua mutation in a Gua/Cyt-lined sequence using Zn2+-cyclen polyacrylamide gel electrophoresis.
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.
Kinoshita
Eiji
E
Department of Functional Molecular Science, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima 734-8553, Japan. kinoeiji@hiroshima-u.ac.jp
Kinoshita-Kikuta
Emiko
E
Yoshimoto
Miki
M
Koike
Tohru
T
D001483:Base Sequence; D000080027:Cyclams; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006571:Heterocyclic Compounds / Q000737:chemistry*; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009942:Organometallic Compounds / Q000737:chemistry*; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D015222:Sodium Channels / Q000235:genetics; D015032:Zinc / Q000737:chemistry*
D000080027:Cyclams; D006571:Heterocyclic Compounds; D009124:Muscle Proteins; D062554:NAV1.5 Voltage-Gated Sodium Channel; D009942:Organometallic Compounds; C568320:SCN5A protein, human; D015222:Sodium Channels; D004247:DNA; C038072:cyclen; D015032:Zinc