Thermodynamics-based rational design of DNA block copolymers for quantitative detection of single-nucleotide polymorphisms by affinity capillary electrophoresis

Ayumi Kimura, Naoki Kanayama, Atsushi Ogawa, Hideaki Shibata, Hideo Nakashita, Tohru Takarada, Mizuo Maeda

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 probes 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 106 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.

Original languageEnglish
Pages (from-to)11425-11433
Number of pages9
JournalAnalytical chemistry
Volume86
Issue number22
DOIs
Publication statusPublished - Nov 18 2014
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry

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