Jo Kyubong, Dhingra Dalia M, Odijk Theo, de Pablo Juan J, Graham Michael D, Runnheim Rod, Forrest Dan, Schwartz David C
Laboratory for Molecular and Computational Genomics, University of Wisconsin, 425 Henry Mall, Madison, WI 53706, USA.
Proc Natl Acad Sci U S A. 2007 Feb 20;104(8):2673-8. doi: 10.1073/pnas.0611151104. Epub 2007 Feb 12.
Molecular confinement offers new routes for arraying large DNA molecules, enabling single-molecule schemes aimed at the acquisition of sequence information. Such schemes can rapidly advance to become platforms capable of genome analysis if elements of a nascent system can be integrated at an early stage of development. Integrated strategies are needed for surmounting the stringent experimental requirements of nanoscale devices regarding fabrication, sample loading, biochemical labeling, and detection. We demonstrate that disposable devices featuring both micro- and nanoscale features can greatly elongate DNA molecules when buffer conditions are controlled to alter DNA stiffness. Furthermore, we present analytical calculations that describe this elongation. We also developed a complementary enzymatic labeling scheme that tags specific sequences on elongated molecules within described nanoslit devices that are imaged via fluorescence resonance energy transfer. Collectively, these developments enable scaleable molecular confinement approaches for genome analysis.
分子受限为排列大型DNA分子提供了新途径，使旨在获取序列信息的单分子方案成为可能。如果新生系统的元件能够在开发的早期阶段进行整合，这样的方案能够迅速发展成为能够进行基因组分析的平台。需要综合策略来克服纳米级设备在制造、样品加载、生化标记和检测方面的严格实验要求。我们证明，当缓冲条件被控制以改变DNA的刚性时，具有微米和纳米级特征的一次性设备能够极大地拉长DNA分子。此外，我们给出了描述这种伸长的分析计算。我们还开发了一种互补的酶标记方案，该方案可标记所述纳米狭缝装置内伸长分子上的特定序列，这些序列通过荧光共振能量转移成像。总的来说，这些进展为基因组分析提供了可扩展的分子受限方法。
