Quantitative single-nucleotide polymorphism analysis in secondary-structured DNA by affinity capillary electrophoresis using a polyethylene glycol–peptide nucleic acid block copolymer

Abstract

To estimate allele frequency of single-nucleotide polymorphisms (SNPs) in pooled DNAs with secondary structures, an affinity capillary electrophoresis was developed using an allele-specific peptide nucleic acid probe modified with polyethylene glycol. This probe disrupted secondary structures of DNA analytes and hybridized to them during electrophoresis. Such DNA-binding capability allowed separation of the folded analytes with a single-base difference within 20min. The feed ratio of the target allele was evaluated by calculating the peak area ratio. The averaged difference between the feed and observed ratio was 1.5%. This method should be of general applicability to quantitative SNP analyses.