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

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.

RAFT-generated polyacrylamide-DNA block copolymers for single-nucleotide polymorphism genotyping by affinity capillary electrophoresis.

Capillary electrophoretic separation of a mixture of 5'-fluorescein isothiocyanate-labeled single-stranded DNA (normal ssDNA) and its single-base-substituted one (mutant ssDNA) was achieved by using a RAFT-generated polyacrylamide-oligodeoxyribonucleotide block copolymer (PAAm-b-ODN) as an affinity polymeric probe. PAAm-b-ODN was synthesized through the Michael addition of thiol-terminated PAAm (PAAm-SH) to 5'-maleimide-modified ODN. PAAm-SH was derived from dithiobenzoate-terminated PAAm prepared via RAFT polymerization. The number-averaged molecular weight (M(n)) and the molecular weight distribution were determined by aqueous size exclusion chromatography. After a capillary tube was filled with the running buffer solution of PAAm-b-ODN, a mixture of normal and mutant ssDNA was subjected to electrophoresis and detected by a laser-induced fluorescent detector. Because the base sequence of PAAm-b-ODN was complementary to part of the mutant ssDNA, including a single-base substitution site, the electrophoretic migration of mutant ssDNA was retarded due to the formation of the equilibrium complex with PAAm-b-ODN. Increasing M(n) of the PAAm segment enhanced this retardation. On the other hand, normal ssDNA was unable to form the complex owing to a single-base mismatch, which was proved by melting curve measurements. The Lineweaver-Burk-type analysis of the mobility of mutant ssDNA revealed that the binding constants for the complexes with different PAAm-b-ODN probes were almost identical to each other. The analysis also demonstrated that the ratio of the hydrodynamic radius of the complex to that of the free mutant ssDNA increased with increasing M(n) of the affinity polymeric probe's PAAm segment. This means that the PAAm segment indirectly provides mutant ssDNA with an additional hydrodynamic friction force via the affinity interaction of the ODN segment. Optimization of the salt concentration of the running buffer and the capillary temperature improved the resolution of the separation. This affinity polymeric probe will be useful for developing a simple and highly reliable single-nucleotide polymorphism genotyping method.

Affinity capillary electrophoretic DNA separation using PEG-oligodeoxyribonucleotide block copolymers: relationship between peak resolution and affinity strength.

Capillary electrophoretic separation of chemically synthesized ssDNA and a single-base-substituted one (normal and mutant ssDNA, respectively) was demonstrated using a PEG-oligodeoxyribonucleotide block copolymer (PEG-b-ODN) as an affinity ligand. When the base sequence of PEG-b-ODN was designed to be complementary to part of normal ssDNA including the base-substituted site, the electrophoretic mobility of normal ssDNA significantly decreased whereas that of mutant ssDNA slightly changed. Resolution of the separation strongly depended on the ODN length of the copolymer, the capillary temperature, and the Mg2+ concentration in the running buffer, indicating that the retardation of migration of normal ssDNA was induced by the reversible hybridization with PEG-b-ODN. It was found that the dissociation constant (K(d)) of the duplex between normal ssDNA and the affinity probe ODN should be smaller than 10(-6) M to achieve the good peak separation. In addition, we calculated the mobility of the complex (mu(C)) between normal ssDNA and PEG-b-ODN using a two-state model. The base sequence of affinity probe ODN appropriate to achieve the sufficient resolution will be predicted on the basis of the mu(C) and K(d )values.

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.

Point mutation assay by affinity capillary electrophoresis using poly(ethylene glycol)-oligodeoxyribonucleotide block copolymers.

An affinity capillary electrophoresis was developed for detecting a point mutation of single-stranded DNA (ssDNA). Poly(ethylene glycol)-oligodeoxyribonucleo-tide block copolymers (PEG-b-ODN) were prepared to use as a novel affinity ligand. Optimization of the analytical conditions, such as the salt concentration of running buffer, the capillary temperature and the base number of the copolymer, gave two distinct peaks for wild-type (WT) and point-mutated ssDNA (MT) on the electropherogram, allowing for the facile discrimination of the single-base difference.