The longest oligos that can be chemically synthesized are considered to be 200-mers. Here, we report direct synthesis of an 800-mer green fluorescent protein gene and a 1,728-mer Φ29 DNA polymerase gene on an automated synthesizer. Key innovations that enabled the breakthrough include conducting the synthesis on a smooth surface rather than within the pores of traditional supports, and the use of the powerful catching-by-polymerization (CBP) method for isolating the full-length oligos from complex mixture. Conducting synthesis on smooth surface not only eliminated the steric hindrance that would otherwise prevent long oligo assembly, but also, surprisingly, drastically reduced synthesis errors. Compared with the benchmark PCR assembly gene synthesis method, the direct long oligo synthesis method has the advanges of higer probability to succeed, less sequence restrictions, and being able to synthesize long oligos containing difficult elements such as unusually stable higher-order structures, long repeats, and site-specific modifications. The method is expected to open doors for various projects in areas such as synthetic biology, gene editing, and protein engineering.

中文翻译：

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长寡核苷酸：直接化学合成具有多达 1,728 个核苷酸的基因


可以化学合成的最长寡核苷酸被认为是 200 聚体。在这里，我们报道了在自动合成仪上直接合成一个 800 聚体绿色荧光蛋白基因和一个 1,728 聚体 Φ29 DNA 聚合酶基因。实现这一突破的关键创新包括在光滑的表面上进行合成，而不是在传统载体的孔内进行合成，以及使用强大的聚合捕获 （CBP） 方法从复杂混合物中分离全长寡核苷酸。在光滑的表面上进行合成不仅消除了阻碍长寡核苷酸组装的空间位阻，而且令人惊讶的是，它大大减少了合成误差。与基准 PCR 组装基因合成方法相比，直接长寡核苷酸合成方法具有成功概率更高、序列限制更少、能够合成含有异常稳定的高阶结构、长重复序列和位点特异性修饰等困难元件的长寡核苷酸。该方法有望为合成生物学、基因编辑和蛋白质工程等领域的各种项目打开大门。