Direct real-time single molecule DNA sequencing

描述（由申请人提供）：我们提出开发一种方法，用于以天然DNA聚合酶的速度和准确度使用天然核苷酸对来自基因组DNA的单个DNA分子进行直接实时测序。 我们将利用DNA复制中使用的真正纳米机器的力量，天然DNA聚合酶。 与用于区分4种碱基类型的纳米孔测序的难以工程化的人造纳米结构不同，DNA聚合酶具有精确的原子分辨率3D结构，并且可以以高保真度和速度合成非常长的DNA分子。 具有校对功能的DNA聚合酶的错误率可以低至百万分之一，并且进行性聚合酶如phi 29 DNA聚合酶可以在一段时间内合成多达100，000个碱基。 从丰富的结构和动力学研究，它是众所周知的，DNA合成的保真度是基于精致的结构互补性和聚合酶蛋白的活性位点和引物/模板/核苷酸复合物之间的许多特定的相互作用。 伴随着特异性相互作用、诱导配合、键断裂/形成和模板易位的动态化学机械或构象变化确保高度准确和有序的碱基配对和掺入。 我们的策略是通过蛋白质工程将传感器工程化到聚合酶的表面（而不是活性位点）上，以监测伴随每种碱基类型的掺入而发生的微妙而独特的构象变化。 小的距离变化（一到几十埃）可以用F“rster共振能量转移（FRET）技术精确测量。 放置在聚合酶上的策略残基中的多个FRET对或网络将用于在真实的时间（比DNA合成速率快10倍）内监测构象变化。 传感器将提供关于聚合酶的动态结构的多参数信息，这很可能将为掺入的每种碱基类型提供独特的签名。 化学修饰，如模板DNA上的甲基化也可能被检测到。 这种方法可以对非常长的DNA分子进行测序，并且可以在几分钟内以高保真度进行测序，并且可以在不到一小时的时间内对人类基因组甚至表观基因组进行测序。 这将真正实现个性化医疗。 公共卫生相关性：我们建议开发一种突破性的DNA测序技术，称为READS基因组技术，用于直接实时单分子测序。 我们的目标是开发新的测序方法，并设计一个用于超快速和低成本人类基因组测序的测序平台，以便可以对个体人类基因组进行常规测序，用于生物医学应用和个性化医疗。

DESCRIPTION (provided by applicant): We propose to develop a method for direct real-time sequencing of single DNA molecules from genomic DNA at the speed and accuracy of the natural DNA polymerases using native nucleotides. We will harness the power of the true nano-machines used in DNA replication, the natural DNA polymerases. Unlike the difficult to engineer man-made nanostructures of nanopore sequencing used to distinguish the 4 base types in close proximity and constant fluctuation, DNA polymerases have precise atomic-resolution 3D structures and can synthesize very long DNA molecules with high fidelity and velocity. The error rate of a DNA polymerase with proof-reading function could be as low as one in a million bases and a processive polymerase such as phi29 DNA polymerase can synthesize up to 100,000 bases in a stretch. From the wealth of structural and kinetics studies, it is well known that the fidelity of DNA synthesis is predicated on the exquisite structural complementarity and the numerous specific interactions between the active site of the polymerase protein and the primer/template/nucleotide complex. The dynamic chemo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formation, and template translocation ensure highly accurate and orderly base pairing and incorporation. Our strategy is to engineer sensors onto the surface (not the active site) of the polymerase by protein engineering to monitor the subtle yet distinct conformational changes accompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can be measured precisely with F"rster resonance energy transfer (FRET) technique. Multiple FRET pairs or networks placed in strategic residues on the polymerase will be used to monitor the conformational changes in real time (10 times faster than the rate of DNA synthesis). The sensors will provide multi-parametric information on the dynamic structures of the polymerase, which very likely will provide a unique signature for each base type incorporated. Chemical modifications such as methylation on the template DNA could also potentially be detected. Such a method could sequence very long DNA molecules and could be sequenced with high fidelity in minutes and a human genome or even epigenome could be sequenced in less than one hour. This will truly enable personalized medicine. PUBLIC HEALTH RELEVANCE: We propose to develop a breakthrough DNA sequencing technology called READS genome technology for direct real-time single molecule sequencing. We aim to develop the new sequencing method and engineer a sequencing platform for ultra-fast and low-cost human genome sequencing so that routine sequencing of individual human genomes can be performed for biomedical applications and personalized medicine.