Department of Physics, University of Washington, Seattle, Washington, United States of America.
PLoS One. 2011;6(10):e25723. doi: 10.1371/journal.pone.0025723. Epub 2011 Oct 4.
Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.
纳米孔测序有可能成为一种快速且低成本的 DNA 测序平台。离子电流通过一个小孔时，会直接映射穿过紧缩区的单链 DNA（ssDNA）的序列。来源于耻垢分枝杆菌的 MspA 孔蛋白具有短而狭窄的通道紧缩区，非常适合纳米孔测序。为了研究 MspA 解析核苷酸的能力，我们使用生物素-链霉亲和素复合物将 ssDNA 固定在孔内。我们发现，腺嘌呤、胞嘧啶、胸腺嘧啶和鸟嘌呤的均聚物在 MspA 中的电流差异比在 α-溶血素中大得多。此外，甲基化胞嘧啶与未甲基化胞嘧啶可区分开来。我们确定，当 ssDNA 固定在 MspA 的紧缩区时，可以检测到其中的单核苷酸取代。利用基因组上的单核苷酸多态性，我们证明可以识别随机 DNA 中的单个核苷酸。我们的结果表明，MspA 具有高信噪比和纳米孔测序设备所需的单核苷酸灵敏度。
