IMMOBILIZATION OF POLYNUCLEOTIDES ON MAGNETIC PARTICLES - FACTORS INFLUENCING HYBRIDIZATION EFFICIENCY

被引:32
作者
DAY, PJR [1 ]
FLORA, PS [1 ]
FOX, JE [1 ]
WALKER, MR [1 ]
机构
[1] UNIV BIRMINGHAM,DEPT BIOCHEM,ALTA BIOSCI,BIRMINGHAM B15 2TJ,W MIDLANDS,ENGLAND
关键词
D O I
10.1042/bj2780735
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 mu-g of immobilized oligonucleotide exceeds that achieved with other forms of immobilization chemistries employing random attachement 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.
引用
收藏
页码:735 / 740
页数:6
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