Laboratoire de Physique Statistique, Ecole Normale Supérieure, Université Pierre et Marie Curie Université Paris 06, Université Paris Diderot, Centre National de la Recherche Scientifique, Paris, France.
Nat Methods. 2012 Mar 11;9(4):367-72. doi: 10.1038/nmeth.1925.
High-throughput, low-cost DNA sequencing has emerged as one of the challenges of the postgenomic era. Here we present the proof of concept for a single-molecule platform that allows DNA identification and sequencing. In contrast to most present methods, our scheme is not based on the detection of the fluorescent nucleotides but on DNA hairpin length. By pulling on magnetic beads tethered by a DNA hairpin to the surface, the molecule can be unzipped. In this open state it can hybridize with complementary oligonucleotides, which transiently block the hairpin rezipping when the pulling force is reduced. By measuring from the surface to the bead of a blocked hairpin, one can determine the position of the hybrid along the molecule with nearly single-base precision. Our approach can be used to identify a DNA fragment of known sequence in a mix of various fragments and to sequence an unknown DNA fragment by hybridization or ligation.
高通量、低成本的 DNA 测序已经成为后基因组时代的挑战之一。在这里，我们提出了一种单分子平台的概念验证，该平台允许进行 DNA 鉴定和测序。与大多数现有方法不同，我们的方案不是基于荧光核苷酸的检测，而是基于 DNA 发夹长度。通过拉动固定在 DNA 发夹上的磁珠，可以将分子拉开。在这种打开状态下，它可以与互补的寡核苷酸杂交，当拉力降低时，寡核苷酸会暂时阻止发夹重新形成。通过测量从表面到被阻塞发夹的珠子，可以以几乎单个碱基的精度确定杂交分子的位置。我们的方法可用于在各种片段的混合物中鉴定已知序列的 DNA 片段，并通过杂交或连接对未知 DNA 片段进行测序。
