IDBR (EAGER): An AFM-Based Instrument for Monitoring DNA Synthesis in Real-Time

摘要该NSF奖支持开发一种能够对人类和其他基因组进行测序的仪器，该仪器具有前所未有的低成本，长读取段，高速度和准确性。为了实现这一目标，原子力显微镜（AFM）的探针将被DNA聚合酶功能化，并且在DNA合成过程中聚合酶的构象扰动将被AFM实时监测。由于不同的核苷酸引起不同的酶的构象扰动，模板DNA的序列将在其通过聚合酶时直接从独特的构象扰动顺序中读出。使用目前市场上的仪器，基因组测序是非常昂贵的，这阻碍了许多应用，如个性化医疗。这些仪器只能获得短读片段，这导致测序后基因组组装的计算量很大。短序列也会给长重复序列的基因组测序带来问题。现有的方法需要通过聚合酶链反应（PCR）扩增样品。由于在样品扩增过程中引入的不准确性，基因组序列在疾病诊断等应用中不够准确。在传统的测序过程中，与许多重要的生物过程相关的表观遗传DNA碱基修饰信息丢失了。提出的基于afm的仪器有望克服这些问题。由于新仪器使用单分子测序，现有技术中DNA样品制备和扩增的成本将降至最低。在许多已知的测序技术中，需要昂贵的试剂。这项新技术可能只需要天然核苷酸，这将进一步降低成本。该仪器用于测序的样本dna不需要PCR扩增，从而避免了现有测序方法在样品制备过程中引入的不准确性。由于DNA合成过程中DNA序列是直接连续读出的，因此测序速度和读取长度将远远超过目前市场上的技术。由于原始样本dna用于测序，并且预计表观遗传修饰的核碱基会引起与未修饰的核碱基不同的聚合酶构象波动，因此预计新仪器将在不丢失任何表观遗传碱基修饰信息的情况下对基因组进行测序。该仪器将对人类健康、食品、能源、环境和国家安全等许多研究领域产生广泛影响，这些领域都需要对人类、动物、植物、细菌、病毒或其他生物的基因组进行测序。除测序外，该仪器还将应用于DNA聚合酶构象动力学研究，提供无法使用已知技术直接获得的数据。使用本项目开发的技术也可以很容易地制作用于研究其他酶的类似仪器。这些仪器将有助于回答有关酶催化的重要基本问题。最初，新的测序服务将通过合作提供给生物研究实验室。之后，这项服务和仪器将商业化地提供给医学、学术和商业实验室。这个项目是高度多学科的。拥有生物学、化学和工程学专业知识的三个研究小组将共同开发该仪器。在此过程中，两名博士后研究员和至少两名博士生将在这些领域获得丰富的研究经验。此外，还将培养3名或3名以上的本科生。这些下一代科学家中的一些人有望帮助仪器和测序技术的商业化。

AbstractThis NSF award supports development of an instrument that is capable of sequencing human and other genomes with unprecedented low cost, long read sections, and high speed and accuracy. To achieve this goal, the probe of an atomic force microscope (AFM) will be functionalized with a DNA polymerase, and the conformational perturbations of the polymerase during DNA synthesis will be monitored by the AFM in real-time. Because different nucleotides cause different conformational perturbations of the enzyme, the sequence of the template DNA will be read out directly from the order of unique conformational perturbations as it travels through the polymerase. Using instruments currently on the market, genome sequencing is highly expensive, which hampers many applications such as personalized medicine. With these instruments, only short read segments can be obtained, which results in high computational efforts in post-sequencing genome assembly. The short reads also cause problems for sequencing genomes that have long repeats. Existing methods require sample amplification by polymerase chain reaction (PCR). Because of inaccuracies introduced during sample amplification, the genome sequences are not accurate enough for applications such as disease diagnosis. During traditional sequencing, information on epigenetic DNA base modifications, which is linked to many important biological processes, is lost. The proposed AFM-based instrument is expected to overcome these problems. Because the new instrument uses single-molecular sequencing, the costs for DNA sample preparation and amplification in existing technologies will be minimized. In many known sequencing technologies, expensive reagents are required. The new technology may only need natural nucleotides, which will further reduce costs. The sample DNAs used for sequencing with the proposed instrument will not need amplification by PCR, thus the inaccuracies introduced during sample preparation in existing sequencing methods will be avoided. Because the sequences of DNAs are read out directly and continuously during DNA synthesis, the sequencing speed and read length will far exceed those of technologies currently on the market. Because original sample DNAs are used for sequencing and epigenetically modified nucleobases are predicted to cause different conformational fluctuations of polymerase from unmodified ones, the new instrument is expected to sequence genomes without losing any epigenetic base modification information.The instrument will have a broad impact on many research areas such as human health, food, energy, environment, and national security, all of which demand sequencing the genomes of human, animals, plants, bacteria, viruses or other organisms. Besides sequencing, the instrument will also find application in DNA polymerase conformational dynamic studies giving data that cannot be obtained directly using known techniques. Similar instruments for studying other enzymes can also be readily made using the technologies developed in this project. These instruments will help to answer important fundamental questions on enzyme catalysis. Initially, the new sequencing service will be provided to biological research labs through collaborations. Later, the service and the instrument will be made commercially available to medical, academic, and commercial labs. The project is highly multidisciplinary. Three research groups that have expertise in biology, chemistry, and engineering will work together to develop the instrument. During this process, two postdoctoral researchers and at least two PhD students will gain extensive research experiences in these fields. In addition, three or more undergraduate students will also be trained. Some of these next generation scientists are expected to help the commercialization of the instrument and sequencing technology.