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. 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.

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. The peak area ratio between WT and MT was in good agreement with the feed ratio. The averaged difference between the feed and observed ratio of MT was determined to be 0.23%, which is lower than that of any other methods. The ACE using the PEG-b-ODN probes in the presence of EOF could be utilized as a facile method for estimating SNP allele frequency in various research fields.

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). Upon applying a voltage, the DNA hairpin migrated slowly compared to a random sequence ssDNA in the presence of the PNA probe. This retardation was induced by strand invasion of the PNA into the DNA hairpin to form a hybridized complex, where the PEG segment received a large amount of hydrodynamic friction during electrophoresis. The binding constant between the PNA probe and the DNA hairpin was easily determined by mobility analysis. This simple method would be potentially beneficial in studying binding behaviors of various artificial nucleotides to natural DNA or RNA.