title: Thermodynamics-based rational design of DNA block copolymers for quantitative detection of single-nucleotide polymorphisms by affinity capillary electrophoresis.
abstract: Diblock copolymers composed of allele-specific oligodeoxyribonucleotide (ODN) and poly(ethylene glycol) (PEG) are used as an affinity probe of free-solution capillary electrophoresis to quantitatively detect single-base substitutions in genetic samples. During electrophoresis, the probe binds strongly to a wild-type single-stranded DNA analyte (WT) through hybridization, while it binds weakly to its single-base-mutated DNA analyte (MT) due to a mismatch. Complex formation with the probe augments the hydrodynamic friction of either analyte, thereby retarding its migration. The difference in affinity strength leads to separation of the WT, MT, and contaminants, including the PCR primers used for sample preparation. The optimal sequence of the probe's ODN segment is rationally determined in such a way that the binding constant between the ODN segment and MT at the capillary temperature is on the order of 10(6) M(-1). The validity of this guideline is verified using various chemically synthesized DNA analytes, as well as those derived from a bacterial genome. The peak area ratio of MT agrees well with its feed ratio, suggesting the prospective use of the present method in SNP allele frequency estimation.
authors.affiliation: Bioengineering Laboratory, RIKEN , 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Ayumi
authors.initials: A
authors.lastname: Kimura
authors.forename: Naoki
authors.initials: N
authors.lastname: Kanayama
authors.forename: Atsushi
authors.initials: A
authors.lastname: Ogawa
authors.forename: Hideaki
authors.initials: H
authors.lastname: Shibata
authors.forename: Hideo
authors.initials: H
authors.lastname: Nakashita
authors.forename: Tohru
authors.initials: T
authors.lastname: Takarada
authors.forename: Mizuo
authors.initials: M
authors.lastname: Maeda
mesh_terms: D004247:DNA / Q000032:analysis* / Q000235:genetics; D019075:Electrophoresis, Capillary; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013816:Thermodynamics
chemical_list: D009838:Oligodeoxyribonucleotides; D011092:Polyethylene Glycols; D004247:DNA