Day P J, Flora P S, Fox J E, Walker M R
University of Birmingham, Department of Clinical Chemistry, Edgbaston, U.K.
Biochem J. 1991 Sep 15;278 ( Pt 3)(Pt 3):735-40. doi: 10.1042/bj2780735.
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.
研究了通过二硫键形成将含有5'-末端硫醇基团的寡核苷酸固定在硫醇末端的顺磁性Biomag磁珠上。结果表明寡核苷酸能以高产率偶联，该连接在90℃下稳定且可逆，并且固定化的寡核苷酸可用于互补杂交，但不能用于非互补杂交。每微克固定化寡核苷酸的特异性杂交能力超过了采用寡核苷酸随机连接和/或特异性末端连接到固体支持物上的其他形式固定化化学方法所达到的能力。在0.2% SDS中孵育可减少DNA在磁珠表面的吸附；其他非特异性封闭剂则无影响。在杂交前将含有固定化寡核苷酸的磁珠在90℃下短暂加热，根据固定化寡核苷酸5'端和硫醇基团3'端是否包含聚（dT）间隔序列，可增加特异性杂交的量。间隔序列长度增加（直至聚（dT16）间隔序列）可使互补序列的杂交呈线性增加。
