On the Feasibility of Using the Intrinsic Fluorescence of Nucleotides for DNA Sequencing.
On the Feasibility of Using the Intrinsic Fluorescence of Nucleotides for DNA Sequencing.
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DOI：
10.1021/jp911229c
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发表时间：
2010-04-29
期刊：
影响因子：
--
通讯作者：
Lakowicz JR
中科院分区：
文献类型：
--
作者：
Chowdhury MH;Ray K;Johnson ML;Gray SK;Pond J;Lakowicz JR
目前，全世界都在努力提高 DNA 测序的速度并降低其成本，国家人类基因组研究所 (NHGRI) 的目标就是以低于 1000 美元的价格对人类基因组进行测序。几种高通量技术正在开发中。其中，使用核酸外切酶的单链测序显得非常有前途。然而，这种方法需要使用外在的高量子产率探针一次完全标记至少两个碱基。这是必要的，因为核苷酸吸收深紫外线 (UV) 并以极低的量子产率发射。因此，DNA 和核苷酸的内在发射并未被用于 DNA 测序。在本文中，我们考虑利用单核苷酸的内在发射来识别单核苷酸的可能性。我们使用时域有限差分 (FDTD) 方法来计算铝纳米颗粒对附近发出紫外线的荧光团的影响。我们发现，当紫外荧光团靠近铝纳米结构时，其辐射功率显着增加。我们表明，在用于激发内在核苷酸发射的波长下，铝颗粒附近的局部激发将会增加。使用 FDTD 模拟，我们表明，典型的 DNA 碱基与适当的铝纳米结构耦合时会产生高度定向的发射。此外，我们提出的实验结果表明，当靠近铝纳米结构时，核苷酸薄膜显示出增强的发射。最后，我们提供了蒙特卡罗模拟，可以预测从碱基固有荧光的发射光谱得出的假定光子数量的高水平碱基识别准确度。我们的结果表明，可以使用增强其固有发射的铝纳米结构来检测和识别单核苷酸。这种能力对于 1000 美元基因组的持续努力非常有价值。
There is presently a worldwide effort to increase the speed and decrease the cost of DNA sequencing as exemplified by the goal of the National Human Genome Research Institute (NHGRI) to sequence a human genome for under $1000. Several high throughput technologies are under development. Among these, single strand sequencing using exonuclease appear very promising. However, this approach requires complete labeling of at least two bases at a time, with extrinsic high quantum yield probes. This is necessary because nucleotides absorb in the deep ultra-violet (UV) and emit with extremely low quantum yields. Hence intrinsic emission from DNA and nucleotides is not being exploited for DNA sequencing. In the present paper we consider the possibility of identifying single nucleotides using their intrinsic emission. We used the finite-difference time-domain (FDTD) method to calculate the effects of aluminum nanoparticles on nearby fluorophores that emit in the UV. We find that the radiated power of UV fluorophores is significantly increased when they are in close proximity to aluminum nanostructures. We show that there will be increased localized excitation near aluminum particles at wavelengths used to excite intrinsic nucleotide emission. Using FDTD simulation we show that a typical DNA base when coupled to appropriate aluminum nanostructures leads to highly directional emission. Additionally we present experimental results showing that a thin film of nucleotides show enhanced emission when in close proximity to aluminum nanostructures. Finally we provide Monte Carlo simulations that predict high levels of base calling accuracy for an assumed number of photons that is derived from the emission spectra of the intrinsic fluorescence of the bases. Our results suggest that single nucleotides can be detected and identified using aluminum nanostructures that enhance their intrinsic emission. This capability would be valuable for the ongoing efforts towards the $1000 genome.
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