Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.定制寡核苷酸集 (Custom-defined oligonucleotide collections) 在合成生物学、靶向测序及细胞遗传学领域具有广泛应用，亦可用于为RNA干扰 (RNA interference)、蛋白质工程 (Protein engineering) 及DNA编码文库 (DNA-encoded libraries) 等技术编码信息。为制备DNA微阵列 (DNA microarrays) 开发的高通量并行DNA合成技术，可生成包含大量不同寡核苷酸的文库，但产物总量极为有限。本研究针对由微阵列合成所得的寡核苷酸集，对比了三种制备大规模单链寡核苷酸文库的方法：第一种方法为将经PCR扩增得到的双链文库中生物素标记的链结合于链霉亲和素包被磁珠后进行碱性解链，该方法几乎不会或完全不会产生非生物素标记的单链DNA (single-stranded DNA, ssDNA)；第二种方法为对PCR扩增文库的磷酸化链进行核酸水解，该方法适用于仅需少量文库的场景；第三种方法则将PCR扩增文库的体外转录 (in vitro transcription) 步骤与将转录所得RNA产物反转录为单链互补DNA (complementary DNA, cDNA) 的步骤相结合，是我们推荐的大规模寡核苷酸文库制备方案。最后，本研究提出一种可去除文库扩增过程中引入的引物结合序列的方法。