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.
Kanayama
Naoki
N
Bioengineering Laboratory, RIKEN, Saitama, Japan. Fax: +81-48-462-4658.
Takarada
Tohru
T
Kimura
Ayumi
A
Shibata
Hideaki
H
Maeda
Mizuo
M
D002846:Chromatography, Affinity / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000737:chemistry; D019075:Electrophoresis, Capillary / Q000379:methods; D008024:Ligands; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D011092:Polyethylene Glycols; D011108:Polymers / Q000737:chemistry*
D008024:Ligands; D009838:Oligodeoxyribonucleotides; D011108:Polymers; D011092:Polyethylene Glycols; D004247:DNA