Evaluation of single-base substitution rate in DNA by affinity capillary electrophoresis.
Capillary electrophoretic separation of 60 mer single-stranded DNA (ssDNA) and a single-base-substituted ssDNA was demonstrated using a size- and composition-controlled poly(ethylene glycol)-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. Under appropriate conditions, PEG-b-ODN and ssDNA with a complementary sequence formed a reversible complex via hybridization during the electrophoresis, while the copolymer did not interact with the single-base-substituted ssDNA. The copolymer's PEG length determined the electrophoretic mobility of the ssDNA; upon formation of the complex, the electrically neutral PEG added hydrodynamic friction to ssDNA. Simultaneously using two types of PEG-b-ODN copolymers whose PEG segments were of different lengths, we achieved the complete separation of the 60 mer ssDNA, its single-base-substituted ssDNA, and impurities. This method was sensitive enough to quantify a slight amount (approximately 1%) of the single-base-substituted ssDNA. The present results suggest that our approach is applicable to quantitative detection of minor genotypes.
Bioengineering Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Naoki
N
Kanayama
Tohru
T
Takarada
Hideaki
H
Shibata
Ayumi
A
Kimura
Mizuo
M
Maeda
D000483:Alleles; D001483:Base Sequence; D003062:Codon; D045303:Cytochromes b / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000235:genetics*; D019075:Electrophoresis, Capillary / Q000379:methods*; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes / Q000138:chemical synthesis / Q000737:chemistry / Q000235:genetics; D011092:Polyethylene Glycols / Q000737:chemistry; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D012680:Sensitivity and Specificity; D013696:Temperature
D003062:Codon; D004277:DNA, Single-Stranded; D008024:Ligands; D015345:Oligonucleotide Probes; D011092:Polyethylene Glycols; D045303:Cytochromes b; D008274:Magnesium