Real-Time DNA Sequencing from Single Polymerase Molecules
Real-Time DNA Sequencing from Single Polymerase Molecules
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DOI：
10.1126/science.1162986
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发表时间：
2009-01-02
期刊：
影响因子：
56.9
通讯作者：
Turner, Stephen
中科院分区：
文献类型：
--
作者：
Eid, John;Fehr, Adrian;Turner, Stephen
我们介绍了从DNA聚合酶获得的单分子实时测序数据，该DNA聚合酶使用四个可区分的荧光标记的脱氧核糖核苷三磷酸盐(DNTPs)进行不间断的模板指导合成。我们用零模波导纳米结构阵列检测了它们在酶作用下掺入不断增长的DNA链的时间顺序，这提供了光学观察体积限制，并能够并行、同时检测数千个单分子测序反应。将荧光团连接到dNTPs的末端磷酸部分，可以在没有空间位阻的情况下连续观察数千个碱基上的DNA合成。数据直接报告聚合酶动力学，揭示了与DNA二级结构相对应的不同聚合状态和停顿位置。将序列数据与已知的参考序列进行比对，以分析每个模板位置的聚合的生物物理参数。从15倍覆盖率的单分子读数中产生共识序列，显示出99.3%的中位准确率，没有超过依赖于荧光团的错误率的系统误差。
We present single- molecule, real- time sequencing data obtained from a DNA polymerase performing uninterrupted template- directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero- mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single- molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single- molecule reads at 15- fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore- dependent error rates.