Nucleotide discrimination with DNA immobilized in the MspA nanopore.
Nucleotide discrimination with DNA immobilized in the MspA nanopore.
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DOI：
10.1371/journal.pone.0025723
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Gundlach JH
中科院分区：
文献类型：
--
作者：
Manrao EA;Derrington IM;Pavlenok M;Niederweis M;Gundlach JH
纳米孔测序有可能成为一种快速、低成本的DNA测序平台。通过小孔的离子电流将直接映射驱动通过收缩的单链DNA（ssDNA）的序列。源自耻垢分枝杆菌的孔蛋白MspA具有理想地适合于纳米孔测序的短而窄的通道收缩。为了研究MspA解析核苷酸的能力，我们使用生物素-NeutrAvidin复合物将ssDNA保持在孔内。我们发现MspA中腺嘌呤、胞嘧啶、胸腺嘧啶和鸟嘌呤的均聚物表现出比α-溶血素大得多的电流差异。此外，甲基化胞嘧啶可与未甲基化胞嘧啶区分开。我们建立了单核苷酸取代的同聚物ssDNA内可以检测到时，举行MspA的收缩。利用基因组单核苷酸多态性，我们证明了随机DNA中的单核苷酸可以被识别。我们的结果表明，MspA具有高信噪比和纳米孔测序装置所需的单核苷酸灵敏度。
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.