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

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 20 min.

Quantitative SNP genotyping by affinity capillary electrophoresis using PEG-oligodeoxyribonucleotide block copolymers with electroosmotic flow.

Quantitative SNP detection was demonstrated with an ACE using a PEG-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as a probe in the presence of an EOF. The probe's PEG segment with large molecular weight and small polydispersity yielded a high resolution in the separation of a chemically synthesized 60-base ssDNA (WT) and its single-base-substituted mutant (MT). A mixture of WT and MT was clearly separated within 10 min by simultaneously using two types of PEG-b-ODN probes whose ODN segments were complementary to WT and MT and whose PEG segments were of different lengths.

Estimation of binding constants of peptide nucleic acid and secondary-structured DNA by affinity capillary electrophoresis.

An affinity capillary electrophoresis method was developed to determine a binding constant between a peptide nucleic acid (PNA) and a hairpin-structured DNA. A diblock copolymer composed of PNA and polyethylene glycol (PEG) was synthesized as a novel affinity probe. The base sequence of the probe's PNA segment was complementary to a hairpin-structured region of a 60-base single-stranded DNA (ssDNA).