Separation of Oligodeoxyribonucleotides Having Single-base Mutation by Affinity Capillary Electrophoresis Using a DNA- polyacrylamide Conjugate

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Gene mutations have been studied in relation to genetic diseases and cancers, and the detection of DNA single-base mutations should be helpful in making medical diagnoses of these disorders. A number of methods such as single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) have been developed for detecting point mutations and single nucleotide polymorphisms (SNPs), but they are still time-consuming and laborious. Rapid, facile and sensitive detection methods are required.In this study, we have developed an affinity capillary electrophoresis for a single-base difference detection of oligodeoxyribonucleotides using a DNA-polyacrylamide conjugate. The DNA-polyacrylamide conjugate was synthesized through copolymerization between acrylamide and ODN macromonomer, which was obtained by coupling reaction between methacryloyloxysuccinimide and ODN having amino-hexyl linker at its 5'-end. The DNA-polyacrylamide conjugates injected into a capillary can serve as pseudo-immobilized affinity ligands because they behave similarly to non-ionic polyacrylamide in terms of migration rate. This novel affinity capillary electrophoresis was successfully applied to separation of oligodeoxyribonucleotides (60 mer) having an oncogene (K-ras) sequence and a version with single-base substituted within 30 minutes. Moreover, sample DNAs obtained from human cells through PCR amplification can be subjected to this separation system.In conclusion, by using DNA-polyAAm conjugate as an affinity ligand we can separate DNAs having a single-base difference on capillary electrophoresis system. This analytical system is promising for the novel gene mutation assay.