Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators
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DOI：
10.1073/pnas.0609513103
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发表时间：
2006-12-26
影响因子：
11.1
通讯作者：
Turro, Nicholas J.
中科院分区：
文献类型：
--
作者：
Ju, Jingyue;Kim, Dae Hyun;Turro, Nicholas J.
在聚合酶反应过程中，固体表面合成（SBS） DNA测序为破译DNA序列提供了一种范式。我们在这里报告这样一个DNA测序系统的建设使用分子工程方法。在这种方法中，四个核基（A、C、G、T）被修饰为可逆终止体，通过将可切割的荧光基团连接到碱基上，并用一个化学上可逆的小片段覆盖3'-OH基团，使它们仍然被DNA聚合酶识别为底物。我们发现烯丙基片段可以成功地作为连接体将荧光基团连接到3'- o -烯丙基修饰的核链上，形成化学上可切割的荧光核核可逆终止体，3'- o -烯丙基- dntps -烯丙基-荧光基团，用于SBS。DNA延伸产物由3′- o -烯丙基- dntps -烯丙基-荧光基团在聚合酶反应中生成，荧光团和3′- o -烯丙基在30 s内通过pd催化的去烯基化在缓冲水溶液中同时去除。这一步双脱烯化反应因此允许重新启动聚合酶反应，并提高SBS效率。通过在DNA芯片和四色荧光扫描仪上使用这类荧光核苷酸类似物，对由均聚物区域组成的DNA模板进行了精确测序。
DNA sequencing by synthesis (SBS) on a solid surface during polymerase reaction offers a paradigm to decipher DNA sequences. We report here the construction of such a DNA sequencing system using molecular engineering approaches. In this approach, four nucleoticles (A, C, G, T) are modified as reversible terminators by attaching a cleavable fluorophore to the base and capping the 3'-OH group with a small chemically reversible moiety so that they are still recognized by DNA polymerase as substrates. We found that an allyl moiety can be used successfully as a linker to tether a fluorophore to 3'-O-allyl-modified nucleoticles, forming chemically cleavable fluorescent nucleoticle reversible terminators, 3'-O-allyl-dNTPs-allyl-fluorophore, for application in SBS. The fluorophore and the 3'-O-allyl group on a DNA extension product, which is generated by incorporating 3'-O-allyl-dNTPs-allyl-fluorophore in a polymerase reaction, are removed simultaneously in 30 s by Pd-catalyzed deallylation in aqueous buffer solution. This one-step dual-deallylation reaction thus allows the reinitiation of the polymerase reaction and increases the SBS efficiency. DNA templates consisting of homopolymer regions were accurately sequenced by using this class of fluorescent nucleotide analogues on a DNA chip and a four-color fluorescent scanner.