3'-O-Modified Nucleotide Reversible Terminators for Pyrosequencing

描述（由申请人提供）：小型设备中的焦磷酸测序已被证明在基因组测序中具有广泛的应用。然而，用天然核苷酸进行焦磷酸测序在准确破译DNA模板的同聚区域方面存在固有的困难。本提案的目标是设计和合成一个可逆核苷酸终止子库来解决这个问题。以下三个目标将被追求：(1)设计和合成四个3'- o -烯丙基标记的核苷酸，对应于A， C， G， T，作为焦磷酸测序的可逆终止子。我们已经以足够的纯度生产了少量的4个3'- o -烯丙基标记的核苷酸，并证明它们可以用于焦磷酸测序，以在DNA的同聚区域产生准确的测序数据。我们将进一步优化酶促条件，利用3'- o -烯丙基标记的核苷酸增加焦磷酸测序的读取长度；(2)设计合成光可切割的3′- o修饰核苷酸可逆终止体，与3′- o -烯丙基标记的核苷酸进行比较，进一步优化焦磷酸测序平台的准确性和读长；(3)利用上述优化的可逆核苷酸终止体，评估不同微米大小的微珠固定与光裂解或化学裂解条件相容的DNA模板进行焦磷酸测序。这里开发的分子工具将有助于优化焦磷酸测序的从头基因组测序，在生物和生物医学应用中具有无与伦比的准确性。

DESCRIPTION (provided by applicant): Pyrosequencing in a miniaturized device has been shown to have wide applications in genome sequencing. However, pyrosequencing with natural nucleotides has inherent difficulty in accurately deciphering the homopolymeric regions of DNA templates. The goal of this proposal is to design and synthesize a library of reversible nucleotide terminators to address this issue. The following three aims will be pursued: (1) Design and synthesis of four 3'-O-allyl-labeled nucleotides corresponding to A, C, G, T, as reversible terminators for pyrosequencing. We have produced small quantity of four 3'-O-allyl-labeled nucleotides in sufficient purity and have demonstrated that they can be used for pyrosequencing to produce accurate sequencing data in homopolymeric regions of DNA. We will further optimize the enzymatic conditions to increase the read length of pyrosequencing using the 3'-O-allyl-labeled nucleotides; (2) Design and synthesis of photocleavable 3'-O-modified nucleotide reversible terminators to compare with the 3'-O-allyl-labeled nucleotides to further optimize the pyrosequencing platform in terms of accuracy and readlength; (3) Evaluation of different micron-sized beads to immobilize the DNA template that are compatible to photocleavage or chemical cleavage conditions to perform pyrosequencing using the optimized set of reversible nucleotide terminators developed above. The molecular tools developed here will facilitate the optimization of pyrosequencing for de novo genome sequencing with unparalleled accuracy for biological and biomedical applications.