Capillary Electrophoretic Discrimination of Single Nucleotide Polymorphisms Using an Oligodeoxyribonucleotide-polyacrylamide Conjugate as a Pseudo-immobilized Affinity Ligand: Optimum Ligand Length Predicted by the Melting Temperature Values

Mishima, Keiko; Takarada, Tohru; Maeda, Mizuo

doi:10.2116/analsci.21.25

Cited by 15 publications

(12 citation statements)

References 21 publications

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“…To discriminate a single-base difference of ssDNA, two electrophoresis-based methodologies have already been developed: the single-strand conformation polymorphism (SSCP) assay [8] and affinity capillary electrophoresis [9,10]. SSCP is based on the difference in folded structures between normal and mutant ssDNA.…”

Section: Introductionmentioning

confidence: 99%

“…To separate normal and mutant ssDNA by affinity capillary electrophoresis, we have utilized ODN-grafted polyacrylamide (PAAm-g-ODN) to serve as a pseudo-immobilized affinity ligand [9,10]. Since the sequence of ODN (6-12 mer) was designed to be complementary to the relatively small area around the mutation site of normal ssDNA, the duplex formation between sample ssDNA and affinity ligand ODN was reversible and in the dynamic equilibrium state.…”

Section: Introductionmentioning

confidence: 99%

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Poly(ethylene glycol)-oligodeoxyribonucleotide block copolymers for affinity capillary electrophoretic separation of single-stranded DNAs with a single-base difference

Kanayama

Takarada

Kimura

et al. 2007

Reactive and Functional Polymers

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Poly(ethylene glycol)-oligodeoxyribonucleotide block copolymers for affinity capillary electrophoretic separation of single-stranded DNAs with a single-base difference

Kanayama

Takarada

Kimura

et al. 2007

Reactive and Functional Polymers

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“…DNA-polyAAm conjugate was synthesized by the same process as reported previously [10]. Briefly, the vinyl-DNA (150 mM) and acrylamide (450 mM) were copolymerized in Milli-Q water at 257C with 0.07% w/v ammonium persulfate and N,N,N',N'-tetramethylethylenediamine.…”

Section: Ace System Using Dna-polyaam Conjugatementioning

confidence: 99%

“…We previously reported a novel method in which a hybridization assay was conducted in CE separation to allow more robust and sensitive detection of known point mutations [8][9][10]. This approach is based on weak affinity capillary electrophoresis (ACE) in which a DNA probe, that was specific for point mutation and conjugated with polyacrylamide (polyAAm), was introduced into a capillary as a pseudoimmobilized affinity ligand.…”

Section: Introductionmentioning

confidence: 99%

Detection of single‐base mutation by affinity capillary electrophoresis using a DNA‐polyacrylamide conjugate

et al. 2005

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We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.

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“…One example is the increased discrimination of single nucleotide polymorphisms afforded by the use of an oligo-DNApolyacrylamide pseudo-immobilized affinity ligand [56]. In this study, the method was used to separate point mutated from normal CYP2C9 DNA.…”

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confidence: 99%

Recent applications of affinity interactions in capillary electrophoresis

2006

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Systems biology depends on a comprehensive assignment and characterization of the interactions of proteins and polypeptides (functional proteomics) and of other classes of biomolecules in a given organism. High-capacity screening methods are in place for ligand capture and interaction screening, but a detailed dynamic characterization of molecular interactions under physiological conditions in efficiently separated mixtures with minimal sample consumption is presently provided only by electrophoretic interaction analysis in capillaries, affinity CE (ACE). This has been realized in different fields of biology and analytical chemistry, and the resulting advances and uses of ACE during the last 2.5 years are covered in this review. Dealing with anything from small divalent metal ions to large supramolecular assemblies, the applications of ACE span from low-affinity binding of broad specificity being exploited in optimizing selectivity, e.g., in enantiomer analysis to miniaturized affinity technologies, e.g., for fast processing immunoassay. Also, approaches that provide detailed quantitative characterization of analyte-ligand interaction for drug, immunoassay, and aptamer development are increasingly important, but various approaches to ACE are more and more generally applied in biological research. In addition, the present overview emphasizes that distinct challenges regarding sensitivity, parallel processing, information-rich detection, interfacing with MS, analyte recovery, and preparative capabilities remain. This will be addressed by future technological improvements that will ensure continuing new applications of ACE in the years to come.

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Capillary Electrophoretic Discrimination of Single Nucleotide Polymorphisms Using an Oligodeoxyribonucleotide-polyacrylamide Conjugate as a Pseudo-immobilized Affinity Ligand: Optimum Ligand Length Predicted by the Melting Temperature Values

Cited by 15 publications

References 21 publications

Poly(ethylene glycol)-oligodeoxyribonucleotide block copolymers for affinity capillary electrophoretic separation of single-stranded DNAs with a single-base difference

Poly(ethylene glycol)-oligodeoxyribonucleotide block copolymers for affinity capillary electrophoretic separation of single-stranded DNAs with a single-base difference

Detection of single‐base mutation by affinity capillary electrophoresis using a DNA‐polyacrylamide conjugate

Recent applications of affinity interactions in capillary electrophoresis

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