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，以开发高通量、低成本的DNA测序，最终目标是为单个基因组支付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碱基的量子产率很低，因此需要这样的非本征探针。我们建议开发金属纳米结构，它将增加本征核苷酸发射的亮度，降低本底，并有效地将发射导向探测器。此外，这些结构将为基地呼叫提供频谱分离。这些影响是可能的，因为 基团与金属中的电子云通过空间近场相互作用，这被称为等离子激元。为了实现单分子本征发射碱基调用，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，设计增强碱基荧光、提供定向发射并适用于高通量测序的几何结构。 具体目的2.测量DNA核苷酸在金属颗粒附近的光物理性质，从而提高量子产率。 具体目标3.确定金属薄膜中纳米孔内或附近的核苷酸的可探测性和最大计数率。 具体目标4.制造和测试为基地调用提供定向发射和光谱分离的金属结构。我们将确定基地呼叫的检测效率和准确性。 与公共健康相关：美国国立卫生研究院制定了以低成本开发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。这个雄心勃勃的目标需要革命性的测序方法，这种方法结合了高通量和高精度。目前正在调查各种化学、物理和光谱方法。这些方法中的大多数使用光谱检测和鉴定碱基或终止的低聚物，这通常需要用外部荧光团标记。外源标记的需要增加了测序的成本和复杂性，并阻碍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顺序地从单链DNA中去除DNA碱基。这种方法需要对待测序链中的所有DNA碱基进行完整标记，这阻止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着对这种方法的高度兴趣。该提案的目标是开发一种利用DNA碱基的本征荧光来检测和鉴定核酸外切酶释放的单核苷酸的方法。本征基区发射极弱。然而，我们已经开发了利用金属纳米结构来提高可见荧光团的量子产率和光稳定性，最近我们证明了我们的方法可以在DNA发射的紫外光波长特性下工作。我们还表明，结构化金属表面可以用来聚焦发射到探测器上，并可以用来抑制背景发射。我们的初步结果表明，有可能增加DNA的本征碱基发射，并以高精度识别碱基。在这个项目中，我们将使用实验和模拟来设计金属结构，以实现单核苷酸的无标记调用。

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.