Department of Physics, Northeastern University, Boston, Massachusetts 02115, USA.
Pacific Biosciences, Menlo Park, California 94025, USA.
Nat Nanotechnol. 2017 Dec;12(12):1169-1175. doi: 10.1038/nnano.2017.176. Epub 2017 Sep 11.
Compared with conventional methods, single-molecule real-time (SMRT) DNA sequencing exhibits longer read lengths than conventional methods, less GC bias, and the ability to read DNA base modifications. However, reading DNA sequence from sub-nanogram quantities is impractical owing to inefficient delivery of DNA molecules into the confines of zero-mode waveguides-zeptolitre optical cavities in which DNA sequencing proceeds. Here, we show that the efficiency of voltage-induced DNA loading into waveguides equipped with nanopores at their floors is five orders of magnitude greater than existing methods. In addition, we find that DNA loading is nearly length-independent, unlike diffusive loading, which is biased towards shorter fragments. We demonstrate here loading and proof-of-principle four-colour sequence readout of a polymerase-bound 20,000-base-pair-long DNA template within seconds from a sub-nanogram input quantity, a step towards low-input DNA sequencing and mammalian epigenomic mapping of native DNA samples.
与传统方法相比，单分子实时 (SMRT) DNA 测序的读长比传统方法长，GC 偏倚更小，并且能够读取 DNA 碱基修饰。然而，由于 DNA 分子进入零模式波导（进行 DNA 测序的zeptolitre 光学腔）的效率低下，从亚纳克数量读取 DNA 序列是不切实际的。在这里，我们表明，配备纳米孔的波导中电压诱导 DNA 加载的效率比现有方法高五个数量级。此外，我们发现 DNA 加载几乎与长度无关，与偏向于较短片段的扩散加载不同。我们在这里证明了在亚纳克输入量的情况下，在几秒钟内将聚合酶结合的 20,000 碱基对长 DNA 模板加载并进行四色序列读出的原理，这是朝着低输入 DNA 测序和天然 DNA 样本的哺乳动物表观基因组图谱迈进的一步。
