Capillary Electrophoretic Discrimination of Single Nucleotide Polymorphisms Using an Oligodeoxyribonucleotide-polyacrylamide Conjugate as a Pseudo-immobilized Affinity Ligand: Optimum Ligand Length Predicted by the Melting Temperature Values

Abstract

We developed a weak-affinity separation system for single-nucleotide polymorphisms (SNPs) based on capillary electrophoresis. In this approach, single-stranded DNA (ssDNA)-polyacrylamide (polyAAm) conjugate was used as a pseudo-immobilized affinity ligand to separate the target DNA, cytochrome P450 2C9 (CYP2C9), and its point mutant. The ligand DNA was designed to be complementary to the normal DNA, and the target DNA was electrophoretically separated by the difference in the affinity with the pseudo-immobilized ligand in the capillary. We showed that the separation efficiency was closely associated with the T_m value of double-stranded DNA (dsDNA) consisting of the target and ligand DNA, which depends on the measurement conditions, such as the base number of the ligand DNA and the concentration of Mg²⁺ in the buffer solution.