With the rapidly decreasing cost of array-based oligo synthesis, large-scale oligo pools offer significant benefits for advanced applications, including gene synthesis, CRISPR-based gene editing, and DNA data storage. Selectively retrieving specific oligos from these complex pools traditionally uses Polymerase Chain Reaction (PCR), in which any selected oligos are exponentially amplified to quickly outnumber non-selected ones. In this case, the number of orthogonal PCR primers is limited due to interactions between them. This lack of specificity presents a serious challenge, particularly for DNA data storage, where the size of an oligo pool (i.e., a DNA database) is orders of magnitude larger than it is for other applications. Although a nested file address system was recently developed to increase the number of accessible files for DNA storage, it requires a more complicated lab protocol and more expensive reagents to achieve high specificity. Instead, we developed a new combinatorial PCR method that outperforms prior work without compromising the fidelity of retrieved material or complicating wet lab processes. Our method quadratically increases the number of accessible oligos while maintaining high specificity. In experiments, we accessed three arbitrarily chosen files from a DNA prototype database that contained 81 different files. Initially comprising only 1% of the original database, the selected files were enriched to over 99.9% using our combinatorial primer method. Our method thus provides a viable path for scaling up DNA data storage systems and has broader utility whenever scientists need access to a specific target oligo and can design their own primer regions.

中文翻译：

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从复杂寡核苷酸库中高效、选择性地检索寡核苷酸的组合 PCR 方法

随着基于阵列的寡核苷酸合成成本迅速降低，大规模寡核苷酸池为高级应用提供了显着优势，包括基因合成、基于 CRISPR 的基因编辑和 DNA 数据存储。从这些复杂的池中选择性地检索特定的寡核苷酸传统上使用聚合酶链反应 (PCR)，其中任何选定的寡核苷酸都会以指数方式放大，以迅速超过未选定的寡核苷酸。在这种情况下，正交 PCR 引物的数量由于它们之间的相互作用而受到限制。这种缺乏特异性提出了严峻的挑战，特别是对于 DNA 数据存储，其中寡核苷酸库（即 DNA 数据库）的大小比其他应用程序大几个数量级。尽管最近开发了一个嵌套文件地址系统来增加 DNA 存储的可访问文件的数量，它需要更复杂的实验室方案和更昂贵的试剂才能实现高特异性。相反，我们开发了一种新的组合 PCR 方法，它在不影响检索材料的保真度或使湿实验室过程复杂化的情况下优于以前的工作。我们的方法在保持高特异性的同时二次增加了可访问寡核苷酸的数量。在实验中，我们从包含 81 个不同文件的 DNA 原型数据库中访问了三个任意选择的文件。最初只包含原始数据库的 1%，使用我们的组合引物方法将所选文件丰富到 99.9% 以上。因此，我们的方法为扩大 DNA 数据存储系统提供了可行的途径，并且在科学家需要访问特定目标寡核苷酸并可以设计自己的引物区域时具有更广泛的用途。