Medžiūnė Justina, Kapustina Žana, Žeimytė Simona, Jakubovska Jevgenija, Sindikevičienė Rūta, Čikotienė Inga, Lubys Arvydas
Department of Research and Development, Thermo Fisher Scientific Baltics, Vilnius, LT-02241, Lithuania.
Faculty of Chemistry and Geosciences, Vilnius University, Vilnius, LT-03225, Lithuania.
Commun Chem. 2022 Mar 16;5(1):34. doi: 10.1038/s42004-022-00649-9.
The ever-growing demand for inexpensive, rapid, and accurate exploration of genomes calls for refinement of existing sequencing techniques. The development of next-generation sequencing (NGS) was a revolutionary milestone in genome analysis. While modified nucleotides already were inherent tools in sequencing and imaging, further modification of nucleotides enabled the expansion into even more diverse applications. Herein we describe the design and synthesis of oligonucleotide-tethered 2',3'-dideoxynucleotide (ddNTP) terminators bearing universal priming sites attached to the nucleobase, as well as their enzymatic incorporation and performance in read-through assays. In the context of NGS library preparation, the incorporation of ddNTP fulfills two requirements at once: the fragmentation step is integrated into the workflow and the obtained fragments are readily labeled by platform-specific adapters. DNA polymerases can incorporate ddNTP nucleotides, as shown by primer extension assays. More importantly, reading through the unnatural linkage during DNA synthesis was demonstrated, with 25-30% efficiency in single-cycle extension.
对低成本、快速且准确的基因组探索的需求不断增长，这就要求改进现有的测序技术。下一代测序（NGS）的发展是基因组分析中的一个革命性里程碑。虽然修饰核苷酸已经是测序和成像中的固有工具，但对核苷酸的进一步修饰使得能够扩展到更多样化的应用中。在此，我们描述了连接有寡核苷酸的2',3'-双脱氧核苷酸（ddNTP）终止子的设计与合成，这些终止子带有连接到核碱基上的通用引物位点，以及它们在通读测定中的酶促掺入和性能。在NGS文库制备的背景下，ddNTP的掺入同时满足两个要求：片段化步骤被整合到工作流程中，并且获得的片段很容易被平台特异性接头标记。如引物延伸测定所示，DNA聚合酶可以掺入ddNTP核苷酸。更重要的是，在DNA合成过程中已证明可以通读非天然连接，单循环延伸效率为25 - 30%。
