A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+-cyclen complex.
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).
Kinoshita-Kikuta
Emiko
E
Department of Functional Molecular Science, Division of Medicinal Chemistry, Graduate School of Biomedical Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8551, Japan.
Kinoshita
Eiji
E
Koike
Tohru
T
D001483:Base Sequence; D001665:Binding Sites; D000080027:Cyclams; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005838:Genotype; D006571:Heterocyclic Compounds / Q000737:chemistry* / Q000494:pharmacology; D006579:Heterozygote; D006801:Humans; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D020641:Polymorphism, Single Nucleotide; D015222:Sodium Channels / Q000235:genetics; D013936:Thymidine / Q000737:chemistry / Q000378:metabolism; D017967:Zinc Compounds / Q000737:chemistry* / Q000494:pharmacology
D000080027:Cyclams; D004277:DNA, Single-Stranded; D006571:Heterocyclic Compounds; D009841:Oligonucleotides; D015222:Sodium Channels; D017967:Zinc Compounds; D004247:DNA; C038072:cyclen; D013936:Thymidine