DNA sequencing by synthesis using 3'-O-azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection.
DNA sequencing by synthesis using 3'-O-azidomethyl nucleotide reversible terminators and surface-enhanced Raman spectroscopic detection.
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使用 3-O-叠氮甲基核苷酸可逆终止子和表面增强拉曼光谱检测合成 DNA 测序。
DOI：
10.1039/c4ra08398a
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发表时间：
2014
期刊：
影响因子：
3.9
通讯作者：
Ju,Jingyue
中科院分区：
文献类型：
--
作者：
Palla,Mirkó;Guo,Wenjing;Shi,Shundi;Li,Zengmin;Wu,Jian;Jockusch,Steffen;Guo,Cheng;Russo,JamesJ;Turro,NicholasJ;Ju,Jingyue
作为一种替代基于荧光的DNA合成测序的方法，我们报告了一种使用叠氮部分(N3)作为合成DNA测序的标记的方法，该部分在2125 cm−1处具有强烈、狭窄和唯一的拉曼位移，其中几乎所有生物分子都是透明的。我们首先证明了4个3‘-O-叠氮甲基核苷酸可逆终止子(3’-O-叠氮甲基-dNTPs)在2125 cM−1处有表面增强拉曼散射，然后以这4个核苷酸类似物为底物，进行了完整的4步反应。我们使用表面增强拉曼光谱监测了叠氮化物特有的拉曼峰的出现，这是由于聚合酶将单个3‘-O-叠氮甲基-dNTP延伸到正在生长的−链中，并随着叠氮标记的断裂而消失，从而允许下一个核苷酸掺入，从而连续确定DNA序列。由于叠氮标记的尺寸较小，3‘-O-叠氮甲基-dNTPs是DNA聚合酶的有效底物。在SBS循环中，天然核苷酸在每次掺入和切割后恢复，产生一条生长的DNA链，不需要任何修饰，也不会阻碍进一步的聚合酶反应。因此，随着叠氮部分的表面增强拉曼光谱的进一步改进，这种方法有可能提供一种有吸引力的替代基于荧光的SBS。
As an alternative to fluorescence-based DNA sequencing by synthesis (SBS), we report here an approach using an azido moiety (N3) that has an intense, narrow and unique Raman shift at 2125 cm−1, where virtually all biological molecules are transparent, as a label for SBS. We first demonstrated that the four 3′-O-azidomethyl nucleotide reversible terminators (3′-O-azidomethyl-dNTPs) displayed surface enhanced Raman scattering (SERS) at 2125 cm−1. Using these 4 nucleotide analogues as substrates, we then performed a complete 4-step SBS reaction. We used SERS to monitor the appearance of the azide-specific Raman peak at 2125 cm−1 as a result of polymerase extension by a single 3′-O-azidomethyl-dNTP into the growing DNA strand and disappearance of this Raman peak with cleavage of the azido label to permit the next nucleotide incorporation, thereby continuously determining the DNA sequence. Due to the small size of the azido label, the 3′-O-azidomethyl-dNTPs are efficient substrates for the DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions. Thus, with further improvements in SERS for the azido moiety, this approach has the potential to provide an attractive alternative to fluorescence-based SBS.
影响因子：
64.8
作者：
Margulies, M;Egholm, M;Rothberg, JM
通讯作者：
Rothberg, JM
用于基因组学和生物医学分析的表面增强拉曼散射 (SERS) 方法和仪器
影响因子：
2.5
作者：
T. Vo‐Dinh;D. Stokes;G. Griffin;M. Volkan;U. Kim;M. Simon
通讯作者：
M. Simon