Methods of synthesizing oligonucleotides with high coupling efficiency (>99.5%) are provided. Methods for purification of synthetic oligonucleotides are also provided. Instrumentation configurations for oligonucleotide synthesis are also provided. Methods of designing and synthesizing polynucleotides are also provided. Polynucleotide design is optimized for subsequent assembly from shorter oligonucleotides. Modifications of phosphoramidite chemistry to improve the subsequent assembly of polynucleotides are provided. The design process also incorporates codon biases into polynucleotides that favor expression in defined hosts. Design and assembly methods are also provided for the efficient synthesis of sets of polynucleotide variants. Software to automate the design and assembly process is also provided.
提供了具有高偶联效率(大于 99.5%)的寡核苷酸合成方法。还提供了纯化合成寡核苷酸的方法。还提供了用于寡核苷酸合成的仪器配置。还提供了设计和合成多核苷酸的方法。对多核苷酸的设计进行了优化,以便随后从较短的寡核苷酸进行组装。提供了对
磷酰胺
化学的修改,以改善多核苷酸的后续组装。设计过程还将密码子偏置纳入多核苷酸,以利于在确定的宿主中表达。此外,还提供了高效合成多核苷酸变体集的设计和组装方法。还提供了设计和组装过程的自动化软件。