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    • 8. 发明授权
    • Solid phase methods for polynucleotide production
    • 用于多核苷酸生产的固相方法
    • US07482119B2
    • 2009-01-27
    • US10965018
    • 2004-10-13
    • Hsing-Yeh ParkerJohn T. Mulligan
    • Hsing-Yeh ParkerJohn T. Mulligan
    • C12Q1/68C12P19/34C07H21/04
    • C12P19/34B01J19/0046B01J2219/00286B01J2219/00308B01J2219/00351B01J2219/00353B01J2219/00389B01J2219/00495B01J2219/00547B01J2219/00585B01J2219/00596B01J2219/00689B01J2219/00698B01J2219/00722C07H21/00
    • Polynucleotides having in excess of 1,000 nucleotides can be prepared using a solid phase synthesis technique. A feature of the technique is the use of a reusable solid support that contains covalently bound oligonucleotide. This covalently bound oligonucleotide is annealed to a bridge oligonucleotide, where the bridge is also annealed to a first oligonucleotide that forms a portion of the target polynucleotide. After the target polynucleotide is synthesized, it can be removed from the solid support under denaturing conditions, and the solid support re-used to prepare additional target polynucleotides. The yield of the target polynucleotide increases when shearing force is applied to the solid support that is linked to the growing oligonucleotide. This shearing force is thought to extend the growing end of the oligonucleotide away from contact with other oligonucleotide bound to the solid support and make that end more accessible to annealing with solution oligonucleotide. The synthesis is conveniently accomplished on a porous frit, where reagents and washing solutions are pumped through the frit.
    • 具有超过1,000个核苷酸的多核苷酸可以使用固相合成技术制备。 该技术的特征是使用含有共价结合的寡核苷酸的可重复使用的固体支持物。 将共价结合的寡核苷酸与桥寡核苷酸退火,其中桥还与形成靶多核苷酸的一部分的第一寡核苷酸退火。 在合成靶多核苷酸之后,可以在变性条件下将其从固体支持物上除去,并且固体支持物再次用于制备另外的靶多核苷酸。 当剪切力施加到与生长的寡核苷酸连接的固体支持物上时,靶多核苷酸的产量增加。 认为这种剪切力使得寡核苷酸的生长末端远离与与固体支持物结合的其它寡核苷酸的接触,并使得该结果更容易用溶液寡核苷酸退火。 合成方便地在多孔玻璃料上完成,其中试剂和洗涤溶液通过玻璃料泵送。