申请人:President and Fellows of Harvard College
公开号:US10774366B2
公开(公告)日:2020-09-15
A method for making a polynucleotide is provided including (a) delivering one or more reaction reagents including an error prone or template independent DNA polymerase, cations and a selected nucleotide to a reaction site including an initiator sequence having a terminal nucleotide for a time period and under conditions capable of covalently adding one or more of the selected nucleotide to the terminal nucleotide at the 3′ end of the initiator such that the selected nucleotide becomes a terminal nucleotide, and (b) determining whether the selected nucleotide has been added to the terminal nucleotide, wherein if the selected nucleotide has not been added to the terminal nucleotide, then repeating step (a) until the selected nucleotide has been added, and (c) repeating steps (a) and (b) until the polynucleotide is formed.
本发明提供了一种制造多核苷酸的方法,包括(a)将一种或多种反应试剂(包括易出错的或与模板无关的DNA聚合酶、阳离子和所选核苷酸)输送到反应位点,该位点包括具有末端核苷酸的启动子序列,反应时间为一段时间,反应条件为能够在启动子的3′末端将一种或多种所选核苷酸共价添加到末端核苷酸上,从而使所选核苷酸成为末端核苷酸;以及(b)确定所选核苷酸是否已添加到末端核苷酸上,其中如果所选核苷酸尚未添加到末端核苷酸上,则重复步骤(a),直到所选核苷酸成为末端核苷酸、和 (b) 确定所选核苷酸是否已添加到末端核苷酸中,其中如果所选核苷酸尚未添加到末端核苷酸中,则重复步骤(a)直到所选核苷酸已添加,和 (c) 重复步骤(a)和(b)直到多核苷酸形成。