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

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.