3'-O-Modified Nucleotide Reversible Terminators for Pyrosequencing

描述（由申请人提供）：小型化装置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，使用天然核苷酸的焦磷酸测序在准确破译DNA模板的均聚区域方面具有固有的困难。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。（1）设计合成4个3 ′-O-烯丙基标记的核苷酸（A、C、G、T）作为焦磷酸测序的可逆终止子。我们已经生产了少量的4种纯度足够的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的均聚区域中产生准确的测序数据。我们将进一步优化酶促条件，以增加使用3 '-O-烯丙基标记的核苷酸的焦磷酸测序的读长;（2）设计和合成可光切割的3'-O-修饰的核苷酸可逆终止子，以与3 '-O-烯丙基标记的核苷酸进行比较，以进一步优化焦磷酸测序平台在准确性和读长方面;（3）评估不同微米尺寸的珠粒以固定DNA模板，所述珠粒与光切割或化学切割条件相容，以使用上文开发的可逆核苷酸终止子的优化组进行焦磷酸测序。这里开发的分子工具将促进焦磷酸测序的从头基因组测序的优化，具有无与伦比的生物和生物医学应用的准确性。

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.