Direct real-time single molecule DNA sequencing

描述(由申请人提供)：我们建议开发一种直接实时测序基因组DNA中单个DNA分子的方法，其速度和准确性与使用天然核苷酸的自然DNA聚合酶相同。我们将利用用于DNA复制的真正纳米机器的力量，即天然DNA聚合酶。DNA聚合酶不同于难以设计的人工纳米孔测序结构，用于区分近距离和恒定波动的四种碱基类型，具有精确的原子分辨三维结构，可以合成高保真和高速度的超长DNA分子。具有校对功能的DNA聚合酶的错误率可能低至百万分之一个碱基，而像phi29 DNA聚合酶这样的过程性聚合酶可以一次合成多达10万个碱基。从丰富的结构和动力学研究中，众所周知，DNA合成的保真度是基于聚合酶蛋白的活性部位与引物/模板/核苷酸复合体之间的精细结构互补性和众多特定的相互作用。伴随着特定相互作用、诱导配对、键断裂/形成和模板移位的动态化学机械或构象变化确保了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在聚合酶的表面(而不是活性部位)设计传感器，以监测伴随着每种碱基类型的加入而产生的微妙但明显的构象变化。用F“rster共振能量转移(FRET)技术可以精确测量微小的距离变化(一到几十埃)。多个FRET对或网络放置在聚合酶上的战略残基中，将用于实时监测构象变化(比DNA合成速度快10倍)。传感器将提供聚合酶动态结构的多参数信息，这很可能为所结合的每一种碱基类型提供唯一的标记。还可能检测到模板DNA上的甲基化等化学修饰。这种方法可以对很长的DNA分子进行测序，并可以在几分钟内高保真地进行测序，而人类基因组甚至表观基因组可以在不到一个小时内完成测序。这将真正使个性化医疗成为可能。 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，并设计一个超快、低成本的人类基因组测序平台，以便为生物医学应用和个性化医学提供单个人类基因组的常规测序。

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.