An integrated system for DNA sequencing by synthesis using novel nucleotide analogues.
An integrated system for DNA sequencing by synthesis using novel nucleotide analogues.
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DOI：
10.1021/ar900255c
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发表时间：
2010-04-20
影响因子：
18.3
通讯作者：
Ju, Jingyue
中科院分区：
文献类型：
--
作者：
Guo, Jia;Yu, Lin;Turro, Nicholas J.;Ju, Jingyue
人类基因组计划已经结束，但其成功完成增加了而不是减少了对高通量 DNA 测序技术的需求。临床筛查个体突变的完整基因组的可能性提供了巨大的好处，无论是对于追求个性化医疗还是揭示基因组对疾病的贡献。桑格测序方法虽然在 30 多年来一直非常高效，但需要一个电泳分离步骤，不幸的是，这仍然是实现经济上可接受的全基因组结果的关键技术障碍。因此，替代测序方法侧重于可以降低成本的创新。 DNA 合成测序 (SBS) 方法作为一种新的测序平台显示出了巨大的前景，最近报道了特别的进展。一般的荧光 SBS 方法包括 (i) 掺入带有荧光报告基因的核苷酸类似物，(ii) 通过荧光发射鉴定掺入的核苷酸，以及 (iii) 荧光团的裂解，以及重新启动聚合酶反应以连续确定序列。在本报告中，我们回顾了 DNA 固定化芯片的构建以及用于 SBS 测序平台的新型核苷酸报告基因的开发。点击化学以其高选择性和偶联效率，被探索用于 DNA 的表面固定。用于 SBS 的第一代 (G-1) 修饰核苷酸具有覆盖 3'-OH 的小化学部分和通过化学可裂解接头连接至碱基的荧光团；该设计确保核苷酸报告基因是聚合酶的良好底物。通过掺入 G-1 修饰核苷酸产生的 DNA 延伸产物上的 3'-加帽部分和荧光团同时被切割，以重新启动聚合酶反应。通过点击化学固定在表面上的 DNA 模板的序列可以通过该芯片-SBS 系统明确识别。第二代（G-2）SBS系统的开发基于这样的概念：添加的核苷酸和引物的结构越接近其天然对应物，聚合酶就越忠实地掺入核苷酸。在这种方法中，聚合酶反应是通过 3' 加帽核苷酸可逆终止子 (NRT) 和可裂解荧光双脱氧核苷酸 (ddNTP) 的组合进行的。通过牺牲少量由 ddNTP 永久终止的引物，大多数由可逆终止子延伸的引物在每个测序循环后恢复为天然引物。我们还开发了3'端核苷酸可逆终止子来解决传统焦磷酸测序中模板均聚物区域的破译问题。 DNA 延伸产物上的 3' 加帽部分会暂时终止聚合酶反应，从而在每个测序循环中仅允许掺入一个核苷酸。因此，使用 3' 加帽的 NRT 可以明确确定同聚区域中的核苷酸数量。已经确定可以通过多种方法将数百万个 DNA 模板固定在芯片表面。因此，将这些高密度 DNA 芯片与本报告中描述的分子级 SBS 方法相结合，有望产生高通量、准确的 DNA 测序系统，在生物研究和医疗保健领域得到广泛应用。
The Human Genome Project has concluded, but its successful completion has increased, rather than decreased, the need for high-throughput DNA sequencing technologies. The possibility of clinically screening a full genome for an individual's mutations offers tremendous benefits, both for pursuing personalized medicine as well as uncovering the genomic contributions to diseases. The Sanger sequencing method—although enormously productive for more than 30 years—requires an electrophoretic separation step that, unfortunately, remains a key technical obstacle for achieving economically acceptable full-genome results. Alternative sequencing approaches thus focus on innovations that can reduce costs. The DNA sequencing by synthesis (SBS) approach has shown great promise as a new sequencing platform, with particular progress reported recently. The general fluorescent SBS approach involves (i) incorporation of nucleotide analogs bearing fluorescent reporters, (ii) identification of the incorporated nucleotide by its fluorescent emissions, and (iii) cleavage of the fluorophore, along with the reinitiation of the polymerase reaction for continuing sequence determination. In this Account, we review the construction of a DNA-immobilized chip and the development of novel nucleotide reporters for the SBS sequencing platform. Click chemistry, with its high selectivity and coupling efficiency, was explored for surface immobilization of DNA. The first generation (G-1) modified nucleotides for SBS feature a small chemical moiety capping the 3′-OH and a fluorophore tethered to the base through a chemically cleavable linker; the design ensures that the nucleotide reporters are good substrates for the polymerase. The 3′-capping moiety and the fluorophore on the DNA extension products, generated by the incorporation of the G-1 modified nucleotides, are cleaved simultaneously to reinitiate the polymerase reaction. The sequence of a DNA template immobilized on a surface via click chemistry is unambiguously identified with this chip-SBS system. The second generation (G-2) SBS system was developed based on the concept that the closer the structures of the added nucleotide and the primer are to their natural counterparts, the more faithfully the polymerase would incorporate the nucleotide. In this approach, the polymerase reaction is performed with the combination of 3′-capped nucleotide reversible terminators (NRTs) and cleavable fluorescent dideoxynucleotides (ddNTPs). By sacrificing a small amount of the primers permanently terminated by ddNTPs, the majority of the primers extended by the reversible terminators are reverted to the natural ones after each sequencing cycle. We have also developed the 3′-capped nucleotide reversible terminators to solve the problem of deciphering the homopolymeric regions of the template in conventional pyrosequencing. The 3′-capping moiety on the DNA extension product temporarily terminates the polymerase reaction, which allows only one nucleotide to be incorporated during each sequencing cycle. Thus, the number of nucleotides in the homopolymeric regions are unambiguously determined using the 3′-capped NRTs. It has been established that millions of DNA templates can be immobilized on a chip surface through a variety of approaches. Therefore, the integration of these high-density DNA chips with the molecular-level SBS approaches described in this Account is expected to generate a high-throughput and accurate DNA sequencing system with wide applications in biological research and health care.
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BTA 是一种新型试剂，用于将 DNA 附着在玻璃上并有效生成固相扩增 DNA 菌落。
影响因子：
14.9
作者：
Fedurco, M;Romieu, A;Williams, S;Lawrence, I;Turcatti, G
通讯作者：
Turcatti, G
DOI：
10.1073/pnas.93.10.4724
发表时间：
1996-05-14
影响因子：
11.1
作者：
Roskey, MT;Juhasz, P;Haff, LA
通讯作者：
Haff, LA
影响因子：
46.9
作者：
Fu, DJ;Tang, K;Köster, H
通讯作者：
Köster, H
影响因子：
46.9
作者：
Drmanac, S;Kita, D;Drmanac, R
通讯作者：
Drmanac, R
影响因子：
56.9
作者：
Kim, Jae Bum;Porreca, Gregory J.;Seidman, J. G.
通讯作者：
Seidman, J. G.