Nanopore Group, Department of Biomolecular Engineering, University of California Santa Cruz, Santa Cruz, California, USA.
Nat Biotechnol. 2012 Feb 14;30(4):344-8. doi: 10.1038/nbt.2147.
An emerging DNA sequencing technique uses protein or solid-state pores to analyze individual strands as they are driven in single-file order past a nanoscale sensor. However, uncontrolled electrophoresis of DNA through these nanopores is too fast for accurate base reads. Here, we describe forward and reverse ratcheting of DNA templates through the α-hemolysin nanopore controlled by phi29 DNA polymerase without the need for active voltage control. DNA strands were ratcheted through the pore at median rates of 2.5-40 nucleotides per second and were examined at one nucleotide spatial precision in real time. Up to 500 molecules were processed at ∼130 molecules per hour through one pore. The probability of a registry error (an insertion or deletion) at individual positions during one pass along the template strand ranged from 10% to 24.5% without optimization. This strategy facilitates multiple reads of individual strands and is transferable to other nanopore devices for implementation of DNA sequence analysis.
一种新兴的 DNA 测序技术使用蛋白质或固态孔，在纳米级传感器上以单分子顺序驱动单个链时对其进行分析。然而，这些纳米孔中的 DNA 不受控制的电泳速度太快，无法进行准确的碱基读取。在这里，我们描述了在不需要主动电压控制的情况下，通过 phi29 DNA 聚合酶控制的α-溶血素纳米孔向前和向后棘轮式驱动 DNA 模板。DNA 链以每秒 2.5-40 个核苷酸的中位数速率通过孔，并以实时 1 个核苷酸的空间精度进行检查。通过一个孔，大约每小时处理 130 个分子中的 500 个分子。在模板链上单个位置的登记错误（插入或缺失）的概率在一次通过中从 10%到 24.5%不等，而无需优化。这种策略有利于对单个链进行多次读取，并可转移到其他纳米孔设备以实现 DNA 序列分析。
