Real-Time DNA Sequencing from Single Polymerase Molecules-CSDN博客

本文链接：https://blog.csdn.net/u010608296/article/details/108916405

Real-Time DNA Sequencing from Single Polymerase Molecules 单聚合酶分子的实时DNA测序

在这里插入图片描述

Abstract

①We detected the temporal order (时间顺序) of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays (零模波导纳米结构阵列), which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions (成千上万的单分子测序反应能够并行、同步检测).
②Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. ( 将荧光团与dNTPs末端磷酸基结合，可以连续观察上千个碱基的DNA合成，而不受空间位阻。这里可能指的是荧光基团的空间位阻不影响DNA的合成)
③Consensus sequences were generated from the single-molecule reads at 15-fold coverage (15倍的测序深度), showing a median accuracy of 99.3%(准确率中位数是99.3%), with no systematic error beyond fluorophore-dependent error rates (除了与荧光基团相关的错误率外，没有系统误差).

Introduction

①Sanger method: This method relies on the low error rate of DNA polymerases (发挥了DNA聚合酶自身的低错误率), but exploits neither their potential for high catalytic rates (高催化效率) nor high processivity (高持续合成能力).
如何理解：大肠杆菌的DNA 聚合酶I三个功能区，5’→3’ DNA聚合酶活性外，还有5’→3’(去除引物)和3’→5’(检查)的外切核酸酶活性。
Klenow fragment，去除5’→3’外切核酸酶活性，在二代测序合成中应用，效率更高。
在这里插入图片描述

②NGS：However, because these methods all gate enzymatic activity, using various termination approaches, they have not yielded longer sequence reads (limited to ~400 nucleotides), nor do they exploit the high intrinsic rates of polymerase-catalyzed DNA synthesis.
③Goal & Challenge：利用DNA聚合酶作为实时测序的引擎，即使用单个碱基对的分辨率直接观察DNA聚合的过程，这一想法早已提出，但一直难以实现。为了充分利用这些酶的固有速度、保真度和加工性，必须同时应对几个技术挑战。
Ⅰ. 聚合酶合成DNA 的速度随机波动
Ⅱ. dNTP的标记不能影响DNA聚合酶的合成
Ⅲ. 需要保持DNA聚合酶的活性，同时抑制标记的dNTP的非特异性吸附
Ⅳ. 最后，需要一种能够准确检测和区分四种不同标记的dNTPs的仪器。

Methods

Technology

▲when a fluorophore is linked to the terminal phosphate moiety (phospholinked), phosphodiester bond formation catalyzed by the DNA polymerase results in release of the fluorophore from the incorporated nucleotide, thus generating natural, unmodified DNA. (当一个荧光团与末端磷酸基连接时，DNA聚合酶催化的磷酸二酯键形成会使荧光团从合并的核苷酸中释放出来，从而生成天然的、未经修饰的DNA)
▲Φ29 DNA polymerase was selected for these studies because it is a stable, single-subunit enzyme with high speed, accuracy, and processivity (稳定的单亚基酶，具有快速、准确和持续合成能力) that efficiently uses phospholinked dNTPs. It is capable of strand-displacement DNA synthesis and has been used in whole-genome amplification, showing minimal sequencing context bias. (链置换复制模式，就是上次提到的一直循环复制的模式，也被广泛用于全基因组扩增)
▲we reported a surface chemistry that enables selective immobilization of DNA polymerase molecules in the detection zone of ZMW nanostructures with high yield. (使DNA聚合酶分子在ZMW纳米结构检测区域的选择性固定化成为可能)
▲可以测到甲基化，甲基化的碱基脉冲的时常和光谱的特征都会变化，所以可以捕捉。

Structure & Pipeline

在这里插入图片描述
(Fig. A) A single molecule of DNA template-bound Φ29 DNA polymerase is immobilized at the bottom of a ZMW。
(Fig. B) Schematic event sequence of the phospholinked dNTP incorporation cycle, with a corresponding expected time trace of detected fluorescence intensity from the ZMW.