DNA Sequencing Using Intricsic Base Fluorescence

描述（由申请人提供）：目前正在进行广泛的努力来开发高通量、低成本的DNA测序，最终目标是单个基因组花费1000美元。一些方法使用物理性质来识别碱基，但大多数方法使用荧光探针作为外部标记。由于DNA碱基的低量子产率，需要这样的外来探针。我们建议开发金属纳米结构，这将增加固有的核苷酸发射的亮度，降低背景，并有效地将发射引向检测器。此外，这些结构将为碱基识别提供光谱分离。这些影响可能是由于 通过空间近场相互作用的基础与电子云的金属，这是所谓的等离子体激元。为了实现单分子固有发射碱基调用，我们提出： 具体目标1。使用理论建模，主要是时域有限差分（FDTD）方法，设计几何结构，增强基础荧光，提供定向发射，并适用于高通量测序。 具体目标2。测量金属颗粒附近的DNA核苷酸的电子物理性质，这增加了量子产率。 具体目标3。确定金属薄膜中纳米孔内或附近核苷酸的可检测性和最大计数率。 具体目标4。制造和测试为基地呼叫提供定向发射和光谱分离的金属结构。我们将确定碱基识别的检测效率和准确性。 公共卫生相关性：美国国立卫生研究院（NIH）制定了一个低成本开发DNA测序的目标。目标是以1000美元的价格对一个人的基因组进行测序。这一雄心勃勃的目标需要革命性的测序方法，将高通量与高准确性相结合。各种各样的化学、物理和光谱方法正在研究之中。这些方法中的大多数使用光谱检测和鉴定碱基或终止的寡聚体，这通常需要用外源荧光团标记。对外源标记的需要增加了测序的成本和复杂性，并且阻止了一些有前途的方法的使用。这些方法之一是使用核酸外切酶从单链DNA中顺序去除DNA碱基。这种方法需要完全标记待测序链中的所有DNA碱基，这阻碍了核酸外切酶测序的广泛使用。尽管如此，大规模并行吞吐量的潜力一直保持在这种方法的高度兴趣。 本研究的目的是建立一种利用DNA碱基的内源荧光检测和鉴定核酸外切酶释放的单核苷酸的方法。本征基发射极弱。 然而，我们已经开发了使用金属纳米结构来增加可见荧光团的量子产率和光稳定性，最近我们表明我们的方法可以在DNA发射的紫外波长特性下工作。我们还表明，结构化的金属表面可用于将发射聚焦到检测器，并可用于抑制背景发射。我们的初步结果表明，这将是可能的，以增加从DNA的固有的碱基发射，并确定与高精度的碱基。在这个项目中，我们将使用实验和模拟来设计用于单核苷酸的无标记调用的金属结构。

DESCRIPTION (provided by applicant): There are extensive ongoing efforts to develop high-throughput low-cost DNA sequencing with the eventual goal of $1000 for an individual genome. Some approaches use physical properties to identify the bases, but most methods use fluorescent probes as extrinsic labels. Such extrinsic probes are needed because of the low quantum yield of the DNA bases. We propose to develop metallic nanostructures which will increase the brightness of intrinsic nucleotide emission, decrease the background, and efficiently direct the emission toward a detector. Additionally, these structures will provide spectral separation for base calling. These effects are possible due to through-space near-field interactions of the bases with electron clouds in the metal, which are called plasmons. To accomplish single-molecule intrinsic emission base calling we propose: Specific Aim 1. Use theoretical modeling, primarily the finite-difference time-domain (FDTD) method, to design geometries which enhance base fluorescence, provide directional emission, and which are practical for high throughput sequencing. Specific Aim 2. Measure the photophysical properties of DNA nucleotides near metal particles which increase the quantum yield. Specific Aim 3. Determine the detectability and maximum count rates for nucleotides in or near nanoholes in metal films. Specific Aim 4. Fabricate and test metallic structures which provide directional emission and spectral separation for base calling. We will determine the detection efficiency and accuracy of the base calling. PUBLIC HEALTH RELEVANCE: The NIH has set a goal of developing DNA sequencing at low cost. The goal is to sequence an individual's genome for $1000. This ambitious goal requires revolutionary approaches to sequencing which combine high throughput with high accuracy. A wide variety of chemical, physical and spectroscopic approaches are under investigation. The majority of these approaches use spectroscopic detection and identification of the bases or terminated oligomers, which typically requires labeling with extrinsic fluorophores. The need for extrinsic labeling increases the cost and complexity of sequencing, and has prevented the use of some promising methods. One of these methods is the use of an exonuclease to sequentially remove DNA bases from a single strand of DNA. This approach requires complete labeling of all the DNA bases in the strand to be sequenced, which has prevented the widespread use of exonuclease sequencing. Nonetheless, the potential for massive parallel throughput has maintained a high interest in this approach. The goal of this proposal is to develop a method to detect and identify single nucleotides released by exonuclease using the intrinsic fluorescence from the DNA bases. Intrinsic base emission is extremely weak. However, we have developed the use of metallic nanostructures to increase the quantum yields and photostability of visible fluorophores and recently we showed that our approach can work at the UV wavelength characteristic of DNA emission. We have also shown that structured metallic surfaces can be used to focus emission towards a detector, and can be used to suppress background emission. Our preliminary results suggest that it will be possible to increase the intrinsic base emission from DNA and to identify the bases with high accuracy. During this project we will use experimentation and simulations to design metallic structures for label-free calling of single nucleotides.