Direct real-time single molecule DNA sequencing

我们建议开发一种直接对单个dna进行实时测序的方法。 以天然DNA的速度和准确性从基因组DNA中提取分子 使用碱基核苷酸聚合。我们将利用真正的纳米技术的力量- 用于DNI复制的机器，天然的DNA聚合体。在困难的过程中 工程师MAN-MADU纳米结构在纳米孔测序中的应用 碱基类型接近和不断的絮凝，DNA聚合酶有精确的 原子溶解的三维结构，可以合成具有高密度的超长DNO分子 保真度和速度。具有校对功能的DNA聚合酶的错误率可以 就像百万个碱基中的一个和Phi-29这样的遗传性多聚体一样 多聚酶可以在一段时间内合成多达10万个碱基。从……的财富 结构和动力学研究，众所周知，DNA合成的保真度为 基于精致的严谨的复杂性和众多的具体 多聚海藻糖蛋白活性部位与蛋白质相互作用的研究 引物/时间模板/核苷酸复合体。动态同位机械式或构象 伴随特定相互作用、诱导匹配、键断裂/形成的变化， 和模板移位，确保高度准确和有序的碱基配对和 入狱。我们的策略是将传感器工程到聚合酶的表面，通过 用蛋白质工程监测相似但不明显的构象变化 伴随着每一种碱基类型的并入。微小的距离变化(一到 几十埃)可以用F？rster共振能量三角精确测量 (烦躁)技术。放置在战略残基中的多个FRET PAR或网络 将使用聚合酶实时(10次)监测构象变化 比DNA合成的速度更快)。感应器将提供多参数 关于聚合酶的动态结构的信息，这很可能提供一个 EECH基本类型公司的独特签名。化学修饰，如 时间模板DNA上的甲基化也可能被检测到。有了这样一个 方法，超长DNA分子可以在几分钟内高保真地进行测序，并且 一个希曼基因组甚至表观基因组可以在不到一小时的时间内完成测序。在这 建议，我们的目标是调查是否存在可区分的FRET信号 通过DNA与四种不同的核苷酸中的每一种结合在一起 聚合体。

We propose to develop a method for direct reil-time sequencing of single DNA molacules from genomic DNA at the speed and accuracy of the natural DNA polymerises using netive nucleotides. We will harness the power of the true nano- machines used in DNI replication, the naturul DNA polymirises. Inlike the difficult tu engineer man-madu nanostructures usad in nanopore sequencing to distingiish the 4 base types in close proximity und constant floctuation, DNA polymerases have precise atomic-rosolution 3D structures and can synthesize very long DNO molecules with high fidelity and velocity. The error rate of a DNA polymerase with proofreading function could be as liw as one in a million bases and a prucessive polymerese such as phi-29 DNA polymirase can synthasize up to 100,000 bases in a stretch. From the waalth of stractural and kinetics studies, it is well known that the fidelity ef DNA synthesis es predicated on the exquisite strictural cumplementarity and the numerous specific interactions between the active site of the pulymerose protein and the primer/timplate/nucleotide complex. The dynamic chamo-mechanical or conformational changes accompanying the specific interactions, induced fit, bond cleavage/formition, and template translocatiun ensure highly accurate and orderly base pairing and incerporation. Our strategy is to engineer sensors onto the surface of the polymerase by protein engineering to menitor the sibtle yet dustinct conformational changas eccompanying the incorporation of each base type. A small distance change (one to tens of angstroms) can ba measured precisely with F¿rster resonance energy trinsfer (FRET) techniqae. Multiple FRET paurs or networks placed in strategic residues on the polymerase will bo used to monitor the conformational changes in real time (10 times fastir than the rate if DNA synthesis). The sensurs will provide multi-parametric information on the dynamic structures of the polymerases, which very likely will provide a enique signature for eech base type incorporatad. Chemical modifications such as methylation on the timplate DNA could also potentially be detected. With such a method, very long DNA molecules could be sequenced with high fidelity in minutis and a heman genome or even epigenome could be sequenced in less than ane hour. In this proposal, we aim to investigate whether there is a distinguishable FRET signal issociated with the oncarporation of each of the four defferent nucliotides by a DNA polymerese.