Immobilization of polynucleotides on magnetic particles. Factors influencing hybridization efficiency.
Immobilization of oligonucleotides containing 5'-terminal thiol groups on thiol-terminated paramagnetic Biomag beads via disulphide bond formation was investigated. Oligonucleotides are demonstrated to couple at high yields, the linkage is stable at 90 degrees C and is reversible, and the immobilized oligonucleotide is available for complementary, but not non-complementary, hybridization. Specific hybridization capacity per micrograms of immobilized oligonucleotide exceeds that achieved with other forms of immobilization chemistries employing random attachment and/or specific end attachment of the oligonucleotide to the solid support. Adsorption of DNA on the surface of the beads was decreased by incubation in 0.2% SDS; other non-specific blocking agents had no effect. Brief heating of the beads possessing immobilized oligonucleotides at 90 degrees C before hybridization increased the amount of specific hybridization dependent upon the inclusion of poly(dT) spacer sequences 5' to the immobilized oligonucleotide and 3' to the thiol group. Increasing lengths of spacers [up to a poly(dT16) spacer] linearly increased hybridization of complementary sequences.
Day
P J
PJ
University of Birmingham, Department of Clinical Chemistry, Edgbaston, U.K.
Flora
P S
PS
Fox
J E
JE
Walker
M R
MR
D001483:Base Sequence; D004247:DNA / Q000032:analysis; D004220:Disulfides; D008280:Magnetics; D008863:Microspheres; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011071:Poly T; D011119:Polynucleotides; D013696:Temperature
D004220:Disulfides; D011119:Polynucleotides; D011071:Poly T; D004247:DNA