Cavendish Laboratory , JJ Thomson Ave, University of Cambridge , Cambridge CB3 0HE , United Kingdom.
Oxford Nanopore Technologies , Gosling Building, Edmund Halley Road , Oxford Science Park OX4 4DQ , United Kingdom.
ACS Sens. 2019 Aug 23;4(8):2065-2072. doi: 10.1021/acssensors.9b00686. Epub 2019 Aug 12.
Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid state nanopore sensors are especially promising for this type of single-molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.
用 dCas9 条码对双链 DNA 进行修饰，以识别特征短序列，为快速、高度特异性分析 DNA 样本提供了一种替代方法，而无需对 DNA 样本进行全序列测序。由于能够分析 DNA 分子离子电流特征中的模式，固态纳米孔传感器特别适合这种类型的单分子传感。在这里，我们系统地展示了使用高度特异性的 dCas9 探针在 DNA 上创建独特条码的方法，这些条码可以使用纳米孔传感器读出。单个 dCas9 探针靶向长达 48 kbp 的 DNA 链的各种位置，并在典型的纳米孔传感高盐条件下有效地进行测量。多个探针结合到同一 DNA 链上的特征靶序列上，形成双和三峰的独特条码。最后，双和三条码用于同时识别细菌 DNA 混合物背景中的两个不同的 DNA 靶标。我们的方法为在复杂样本中进行多重 DNA 传感应用的快速、多功能检测奠定了基础。
