Continuous base identification for single-molecule nanopore DNA sequencing
Continuous base identification for single-molecule nanopore DNA sequencing
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DOI：
10.1038/nnano.2009.12
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发表时间：
2009-04-01
影响因子：
38.3
通讯作者：
Bayley, Hagan
中科院分区：
文献类型：
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作者：
Clarke, James;Wu, Hai-Chen;Bayley, Hagan
不需要荧光标记的单分子 DNA 测序方法可以降低成本并提高测序速度。核酸外切酶可用于从 DNA 上切割单个核苷酸分子，当与适当的检测系统结合时，可以以正确的顺序识别这些核苷酸。在这里，我们证明具有共价连接接头分子的蛋白质纳米孔可以连续识别未标记的核苷W-单磷酸分子，平均准确度为99.8%。甲基化胞嘧啶还可与四种标准 DNA 碱基区分开来：鸟嘌呤、腺嘌呤、胸腺嘧啶和胞嘧啶。操作条件与核酸外切酶兼容，动力学数据表明核苷酸通过纳米孔易位的可能性很高，因此不会被记录两次。这种高度准确的工具适合集成到核酸测序和表观遗传修饰分析系统中。
A single-molecule method for sequencing DNA that does not require fluorescent labelling could reduce costs and increase sequencing speeds. An exonuclease enzyme might be used to cleave individual nucleotide molecules from the DNA, and when coupled to an appropriate detection system, these nucleotides could be identified in the correct order. Here, we show that a protein nanopore with a covalently attached adapter molecule can continuously identify unlabelled nucleoside W-monophosphate molecules with accuracies averaging 99.8%. Methylated cytosine can also be distinguished from the four standard DNA bases: guanine, adenine, thymine and cytosine. The operating conditions are compatible with the exonuclease, and the kinetic data show that the nucleotides have a high probability of translocation through the nanopore and, therefore, of not being registered twice. This highly accurate tool is suitable for integration into a system for sequencing nucleic acids and for analysing epigenetic modifications.